SBE APTAMERS FOR TREATING IL-17a RELATED DISEASES AND CONDITIONS

ABSTRACT

Provided herein are compositions, systems, kits, and methods for treating IL-17a related diseases and conditions using an SBE nucleic acid sequence that binds a SEFIR domain of an ACT1 protein.

The present application is a continuation of U.S. patent applicationSer. No. 16/632,757, filed Jan. 21, 2020, which is a § 371 nationalentry application PCT/US2018/042899, filed Jul. 19, 2018, which claimspriority to U.S. Provisional application 62/535,559, filed Jul. 21,2017, each of which is herein incorporated by reference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under grant nos.HL103453, CA062220, HL029582 awarded by the National Institutes ofHealth and RG5130A2/1 awarded by National Multiple Sclerosis Society.The government has certain rights in the invention.

SEQUENCE LISTING

The text of the computer readable sequence listing filed herewith,titled “34963-304_SEQUENCE_LISTING_ST25”, created Jun. 6, 2022, having afile size of 31,801 bytes, is hereby incorporated by reference in itsentirety.

FIELD

Provided herein are compositions, systems, kits, and methods fortreating IL-17a related diseases and conditions using an SBE nucleicacid sequence that binds a SEFIR domain of an ACT1 protein.

BACKGROUND

Interleukin 17 (IL-17, also known as IL-17A) is a key signature cytokineof Th17 cells and is also produced by innate immune cells (Harrington etal., 2005; Park et al., 2005; Cua and Tato, 2010). While IL-17 isrequired for host defense against extracellular microorganisms (Cho etal., 2010; Conti et al., 2009; Kolls and Khader, 2010; Milner andHolland, 2013), IL-17 plays a critical role in the pathogenesis ofautoimmune and inflammatory diseases, including psoriasis, rheumatoidarthritis, multiple sclerosis, and asthma (Swaidani et al., 2009; Kanget al., 2010; Bulek et al., 2011; Patel et al., 2013).

IL-17 signals through a heterodimeric receptor complex composed ofIL-17RA and IL-17RC (Shen and Gaffen, 2008; Toy et al., 2006). BothIL-17RA and IL-17RC belong to a SEFIR protein family, which is definedby the presence of a conserved cytoplasmic SEFIR domain (Novatchkova etal., 2003). Act1 (also known as CIKS) is an essential component in IL-17signaling and also a member of the SEFIR protein family (Li et al.,2000; Chang et al., 2006; Qian et al., 2007). Upon IL-17 stimulation,Act1 is recruited to IL-17R through a SEFIR-dependent interaction. Act1in turn engages members of the TRAF family, activating NFkB, C/EBP, andMAPK pathways. IL-17-Act1-mediated signaling results in transcription ofpro inflammatory and neutrophil-mobilizing cytokines and chemokines,including CXCL1, TNF, IL-6 and GM-CSF(Gu et al., 2013).

While IL-17 activates gene transcription of cytokines and chemokines, itis equally important for IL-17 to stabilize otherwise unstable mRNAs forthe induction of the pro-inflammatory genes. Cytokine and chemokinemRNAs have short half-lives because of conserved cis-elements, includingAU-rich elements (AREs) and stem-loop (SL) structures in their 3′ UTRs(Leppek et al., 2013; Stoecklin et al., 2006a). The AREs within the 3′UTR can be recognized by RNA binding proteins (including TTP, AUF1, KSRPand SF2) that function to mediate the sequential deadenylation,decapping, and ultimately exonucleolytic degradation of the RNA(Schoenberg and Maquat, 2012; Stumpo et al., 2010). Notably, P-bodiesare sites of mRNA degradation. Stress granules form in response tostress, are sites of RNA triage, and can deliver mRNAs to P-bodies fordecay. Recent studies have reported that SLs present in immune-relatedmRNAs, including TNF and IL-6, are destabilized by RNA binding proteinsRoquin and Regnase-1. Roquin destabilizes translationally inactive mRNAsthat are accumulated in processing-bodies (P-bodies) and stressgranules. Regnase-1 specifically cleaves and degrades translationallyactive mRNAs bound to polysomes (Mino et al., 2015). Although multiplemRNA destabilizing mechanisms have been discovered, there is still asignificant gap in knowledge as to how the mRNAs of inflammatory genesare selectively stabilized and successfully translated in response to aninflammatory stimulus, e.g. IL-17 stimulation.

SUMMARY

Provided herein are compositions, systems, kits, and methods fortreating IL-17a related diseases and conditions using an SBE nucleicacid sequence that binds a SEFIR domain of an ACT1 protein.

In some embodiments, provided herein are methods of treating an IL-17arelated disease or condition comprising: treating a subject with anIL-17a related disorder or condition with a composition, wherein thecomposition comprises a first nucleic acid sequence, wherein the firstnucleic acid sequence comprises an SBE nucleic acid sequence that bindsa SEFIR domain of an ACT1 protein. In certain embodiments, the treatingreduces or eliminates at least one symptom related to the IL-17a relateddisease or condition. In further embodiments, the IL-17a related diseaseis selected from the group consisting of: psoriasis, chronic plaque,asthma, an autoimmune disease, an inflammatory condition, rheumatoidarthritis, and multiple sclerosis. In particular embodiments, thesubject is a human or other mammal.

In additional embodiments, provided herein are compositions comprising afirst nucleic acid sequence, wherein the first nucleic acid sequencecomprises an SBE nucleic acid sequence that binds a SEFIR domain of anACT1 protein, and wherein the first nucleic acid sequence comprisesmodified bases to improve stability in vivo.

In particular embodiments, provided herein are compositions comprising afirst nucleic acid sequence, wherein the first nucleic acid sequencecomprises an SBE nucleic acid sequence that binds a SEFIR domain of anACT1 protein, and wherein the SBE nucleic acid sequence comprises asequence shown in SEQ ID NO:1, 2, or 103, but which is not naturallyoccurring.

In further embodiments, at least a portion of the SBE nucleic acidsequence is from a gene selected from CXCL1, GM-CSF, IL-6, and TNF. Infurther embodiments, the SBE nucleic acid sequence comprises a sequenceshown in SEQ ID NOs:1-2, 49-61, 88-94, and 99-129. In regard to SEQ IDNOS:1 and 2, the N's in these sequences are independently selected fromA, G, C, T, U, as well as modified and non-canonical nucleotide bases.Candidate sequences that are constructed based on the variability in SEQID NOS:1, 2, and 103 may be screened in the same assays employed inExample 1 to determine if they bind a SEFIR domain of an ACT1 protein.

In certain embodiments, the SBE nucleic acid sequence comprises RNAbases (e.g., all or most of the SBE nucleic acid sequence is composed ofRNA bases). In other embodiments, the SBE nucleic acid sequencecomprises DNA bases (e.g., all or most of the SBE nucleic acid sequenceis composed of DNA bases). In some embodiments, the SBE nucleic acidsequence comprises, consist of, or consists essentially of: nucleotides810-857 of the CXCL1 gene, ii) nucleotides 830-856 of the CXCL1 gene, oriii) nucleotides 800-835 of the CXCL1 gene. In further embodiments, theSBE nucleic acid sequence is from a human gene, or has 1 or 2conservative amino acid substitutions compared to the SBE sequence froma human gene.

In some embodiments, the ACT1 protein is human ACT1 protein or othermammalian ACT1 protein. In further embodiments, the first nucleic acidsequence is between 12 and 70 nucleotides in length. In additionalembodiments, the first nucleic acid is present in the composition at alevel that is therapeutic when administered to a subject with an IL-17arelated disease or condition (e.g., for administration to a human).

DESCRIPTION OF THE FIGURES

FIGS. 1A-I. IL-17 induces distinct Act1-RNPs in the nucleus andcytoplasmic granules. A. Confocal imaging of Act1-GFP in HeLa Tet-Onstable cell line induced by doxycycline for 24 hours followed by IL-17stimulation for 0, 1 or 2 hours. Nuclei stained with DAPI. Bar graphshows the percentages of cells that display Act1 localization in thecytoplasmic granules or nuclear localization with and without IL-17stimulation. The quantification is based on analysis of over 50expressing cells with and without IL-17 stimulation followed bytwo-tailed Student's t-test. Data represent mean±SD; *, p<0.05, **,p<0.01. B. Confocal imaging of HeLa cells co-transfected with GFP- orRPF-tagged expression constructs as indicated. Nuclei stained with DAPI.Bar graph shows the percentages of cells with co-localization of the twoexpressing proteins (Act1/Dcp1; Act1/TIA1 and Dcp1/IRAK1). Thequantification is based on the analysis of over 50 expressing cells,followed by two-tailed Student's t-test. Data represent mean±SD; **,p<0.01. C. In situ PLA in HeLa cells untreated or treated with IL-17 (50ng/ml) for 1 hour. Anti-Act1 and Anti-Dcp1 were used as primary mouseantibodies followed by PLA probes. The red dots indicate the interactionof endogenous Act1 and Dcp1. Nuclei stained with DAPI in blue. Bar graphshows the percentages of PLA positive cells with and without IL-17stimulation. The quantification is based on the analysis of 50 cellswith and without IL-17 stimulation, followed by two-tailed Student'st-test. Data represent mean±SD; **, p<0.01. D. Lysates from HeLa cellsstimulated with IL-17 (50 ng/ml) for the indicated time points, eitherleft untreated or treated with RNase A, were immunoprecipitated (IP)using anti-Act1 followed by Western blot analysis with the indicatedantibodies. All above data are representative of at least twoindependent experiments. E. Confocal imaging of HeLa cellsco-transfected with GFP- and RPF-tagged expression constructs asindicated. Nuclei stained with DAPI. Bar graph shows the percentages ofcells with co-localization of the two expressing proteins (Act1/Dcp1;Dcp1/HuR and Dcp1/SF2). The quantification is based on the analysis ofover 50 expressing cells, followed by two-tailed Student's t-test. Datarepresent mean±SD; **, p<0.01. F. In situ PLA assay in HeLa cellstransfected with the indicated expression constructs. Primary mouseantibodies against Flag-tag and rabbit antibodies against HA-tag wereused. The red dots indicate the interaction of the two proteinsincluding Dcp1-Flag/Act1-HA, IL-17-RA-Flag/Act1-HA, HuR-Flag/Act1-HA andSF2-Flag/Act1-HA. The co-transfection of IRAK1-Flag and Act1-HA was usedas a negative control for PLA assay. Nuclei stained with DAPI. Theexpression levels of the transfected constructs were also analyzed bywestern analyses shown below the imaging data. G. Bar graph shows thepercentages of PLA positive cells shown in F. The quantification isbased on the analysis of 50 cells, followed by two-tailed Student'st-test. Data represent mean±SD; **, p<0.01. H. Bar graph shows thepercentages of cells with co-localization of Dcp1 with full-length Act1or Act1 deletion mutants. The quantification is based on the analysis ofover 50 expressing cells, followed by two-tailed Student's t-test. Datarepresent mean±SD; *, p<0.05, **, p<0.01. I. Act1−/−MEFs reconstitutedby retroviral infection with either FLAG-tagged mouse wild-type Act1(WT) or SEFIR1 deletion mutant were treated with IL-17 (50 ng/ml) forthe indicated times. The cell lysates were immunoprecipitated (IP) withanti-FLAG followed by Western blot analysis using antibodies asindicated. Data are representative of two independent experiments.

FIGS. 2A-K. Act1 directly binds to the CXCL1 3′UTR through the SEFIRdomain A. The structure model of Act1-SEFIR was built by SWISS-Model,using the crystal structure of IL-17RA-SEFIR as the template (Zhang etal., 2014) (PDBcode:4NUX). Surface representation of Act1-SEFIR (a) andIL-17RA-SEFIR (c) are shaded by electrostatic potential. Key secondarystructure elements and residues of Act1-SEFIR (b) and IL-17RA-SEFIR (d)are labeled in cartoon representation. B. Binding of purifiedrecombinant His-MBP-Act1 SEFIR (residues 379-538), His-IL-17RA SEFIR(residues 378-536) and His-MBP to the CXCL1 3′UTR (residues 720-940) andGpx4 3′UTR (residues 775-962) was examined in vitro by REMSA.Radiolabeled RNA probes were incubated with increasing amounts ofprotein as indicated. Free RNA was separated from RNA-protein complexesby native PAGE. Graph indicates the apparent Kd of CXCL1 3′UTR (720-940,CXCL1 220 as shown in FIG. 3A), which was calculated as theconcentration of Act1-SEFIR protein required to achieve 50% binding ofthe RNA. Data are representative of three independent experiments (meanand s.d.). C. Binding of purified recombinant His-MBP-Act1 SEFIR andΔSEFIR1, ΔSEFIR2, ΔSEFIR3, ΔSEFIR4 and ΔSEFIR5 to the CXCL1 3′UTR (nt720-940) as described in (B). D. Table shows the apparent Kds of CXCL13′UTR (720-940, CXCL1 220) binding to the indicated proteins in C. Dataare representative of three independent experiments (mean and s.d.). E.FLAG-tagged mouse Act1 or SEFIR deletion mutants were transfected intoHela cells with V5-tagged IL-17RA. The cell lysates of the transfectedcells were immunoprecipated (IP) using anti-V5 followed by Western blotanalyses using the indicated antibodies. Data are representative of twoindependent experiments. F. Act1−/− MEFs reconstituted by retroviralinfection with either FLAG-tagged mouse wild-type Act1 (WT) or SEFIRdeletion mutants were treated with IL-17A (50 ng/ml) for the indicatedtimes. The cell lysates were analyzed by Western blotting with theindicated antibodies. G. Act1−/− MEFs reconstituted by retroviralinfection with either FLAG-tagged mouse wild-type Act1 (WT) or SEFIRdeletion mutants were either left untreated or stimulated with IL-17A(50 ng/ml) for 0, 2, 6 or 24 hours. The mRNA and protein levels wereanalyzed by RT-PCR (Top) and Elisa (Bottom), respectively. Data arerepresentative of two independent experiments (mean and s.d., *, p<0.05,**, p<0.01 by Student's t-test). H. Wild-type Act1, SEFIR deletionmutants or empty vector were co-transfected into HeLa Tet-Off cells withpTRE2 (CXCL1 220, nt 720-940, Datta et al., 2010), followed by treatmentwith doxycycline. RNA samples were prepared from the transfected cellsand subjected to RNA blot analysis. Graph indicates the CXCL1 mRNAlevels normalized to GAPDH and presented as half-life. Data arerepresentative of two independent experiments. (mean and s.d., *, p<0.05by Student's t-test). I. Binding of purified recombinant His-MBP-Act1SEFIR and ΔSEFIR1 to the CXCL1 3′UTR (720-940), TNF 3′UTR (1362-1507)and GM-CSF 3′UTR (513-785) was examined by REMSA as in (B). Tableindicates the apparent Kds of CXCL1 3′UTR (720-940), TNF 3′UTR(1362-1507) and GM-CSF 3′UTR (513-785), which were calculated as theconcentration of His-MBP-Act1 SEFIR and ΔSEFIR1 required to achieve 50%binding of the RNA (quantification see Material and Methods). Data arerepresentative of three independent experiments (mean and s.d.). J.Act1−/−MEFs reconstituted with flag-Act1 were pre-treated with TNF for 1hour and then treated with IL-17 for 0 and 60 min followed by RNAimmunoprecipitation with anti-FLAG and RT-PCR analyses of the indicatedmRNAs. The presented are the relative values to levels from IgGimmunoprecipitation. K. RNA decay in Act1−/−MEFs reconstituted byretroviral infection with either FLAG-tagged mouse wild-type Act1 (WT)or SEFIR deletion mutants pre-treated with TNF for 1 hour and thentreated with Actinomycin D alone (NT) or in the presence of IL-17A (50ng/ml). The indicated mRNA levels were normalized to GAPDH and presentedas decay over time (left) and half-life (right). Data are representativeof two independent experiments. (mean and s.d., *, p<0.05 by Student'st-test).

FIGS. 3A-G. Act1 SEFIR binds to a stem-loop structure in CXCL1 3′UTR. A.Schematic representation of the mouse CXCL1 3′UTR together with analignment of the region containing conserved stem-loops in the CXCL13′UTRs from indicated species. This alignment includes mouse (SEQ IDNO:81), rat (SEQ ID NO:82), human (SEQ ID NO:83), chimpanzee (SEQ IDNO:84), hamster (SEQ ID NO:85), horse (SEQ ID NO:86) and guinea pig (SEQID NO:87). The nucleotides forming conserved stem-loops are indicatedwith shaded boxes. Mouse CXCL1 sequence is numbered with respect tofirst nucleotide of the UTR. B. REMSA was performed to examine thebinding of purified recombinant His-MBP-Act1 SEFIR to the serialdeletion mutants from both ends of CXCL1 220 (720-940) (as indicated inA). Table shows the apparent Kds of the serial deletion mutants CXCL1120 (780-900), CXCL1 110 (780-890), CXCL1 90 (790-880), CXCL1 80(800-880), CXCL1 70 (800-870), which were calculated as theconcentration of His-MBP-Act1 SEFIR required to achieve 50% binding ofthe RNA (quantification see Material and Methods). Data arerepresentative of three independent experiments (mean and s.d.). C.Binding of purified recombinant His-MBP-Act1 SEFIR to SEFIR BindingElement (SBE WT, CXCL1 47 as indicated in A) and stem-loop B andstem-loop C disruption mutants (SBE mutant B and SBE mutant C, Table 2in the Material and Methods) were examined by REMSA. D. Real-time PCRanalysis of CXCL1 and GAPDH mRNA in HeLa Tet-Off cells transfected withpTRE2 (CXCL1 220, nt 720-940, Datta et al., 2010), stem-loop B orstem-loop C disruption mutants (SBE mutant B and SBE mutant C, Table 2),were treated with doxycycline alone or together with IL-17 (50 ng/ml)for 0, 45 or 90 min. CXCL1 mRNA levels were normalized to GAPDH andpresented as half-life. Data are representative of 2 independentexperiments (mean and s.d., *, p<0.05 by Student's t-test). E. The 5′end-labeled CXCL1 SBE was incubated in the absence or presence of Act1.The reactions were then partially digested with RNase T1, A, or V1 asindicated. The products were analyzed by denaturing gel electrophoresis.The sequencing (G and C+U) and alkali ladders are shown in the leftlanes. The numbers to the left of the gel indicate the positions ofnucleotides using the numbering in (F). The bands above C46 are resultedfrom the remaining sequence in plasmid template after restriction enzymecutting. The different regions of the SBE are indicated. The gel shownis a representative example from 3 independent experiments. The redboxes indicate protected nucleotides in the SBE that were consistentlyobserved. F. The structure of the SEFIR binding element from the mouseCXCL1 mRNA is shown (SEQ ID NO:80). Stars indicate nucleotides that areprotected from cleavage by RNase A and V1. G. Binding of purifiedrecombinant His-MBP-Act1 SEFIR to the Stem-loop C and mutants wereexamined by REMSA.

FIGS. 4A-I. Act1-RNA binding to 3′UTR inhibits decapping throughTBK1-mediated phosphorylation of Dcp1. A. (a-d) Cap (m7GDP) labeledreporter RNAs [CXCL1 220 (a) and mutant without stem-loop C (CXCL1220-stem-loop C mutant, c)] were subjected to decapping assay usingpurified Dcp1/Dcp2 with increasing amounts of purified Act1 WT or Act1ΔSEFIR1 in the presence or absence of TBK1 inhibitor (MRT67307). Thepurification of proteins were described in Material and Methods. EDTAand boiling were included as negative controls to inactivate Dcp1/Dcp2enzymatic activity. Radiograms were quantified by densitometry. B. Graphindicates the decapping activity calculated by quantifying the releasedCap as a percentage of the amount of Cap catalyzed by purified Dcp1/2.Data are representative of three independent experiments (mean ands.d.). C. Cell lysates from Act1−/−MEFs with and without reconstitutionof wild-type Act1 (WT and Act1 KO) were immunoprecipitated (IP) withanti-TBK1 followed by Western blot analysis using antibodies asindicated. Data are representative of two independent experiments. D.Cell lysates from untreated and IL-17-treated HeLa cells wereimmunoprecipitated (IP) with anti-Act1 followed by Westernblot analysisusing antibodies as indicated. Data are representative of twoindependent experiments. E. Western blot analysis of lysates fromuntreated or IL-17A treated mouse kidney epithelial cells (from TBKf/fKKi−/− and TBKf/f IKKi+/− mice) infected with a GFP adenovirus (CTRL) orCre-GFP adenovirus (Cre). Data are representative of two independentexperiments. Western blots were quantified by densitometry using ImageJ.F. In vitro kinase assay of Dcp1 by recombinant TBK1 usingDcp1-immunoprecipitates from HeLa cells transfected with FLAG-taggedDcp1. G. V5-tagged wild-type Dcp1 (left panel) or S315A mutant (rightpanel) were transfected into HeLa cells with HA-tagged TBK1 and/orFLAG-tagged Dcp2. The cell lysates were immunoprecipitated (IP) usinganti-FLAG followed by Western blot analysis using the indicatedantibodies. Data are representative of two independent experiments.Western blots were quantified by densitometry using ImageJ. H. Celllysates from untreated and IL-17-treated MEFs with or without thepresence of TAK1 inhibitor (MRT67307) were immunoprecipitated (IP) withanti-Act1 followed by Western blot analysis using antibodies asindicated. Data are representative of two independent experiments.Western blots were quantified by densitometry using ImageJ. I. The samecells as described in (E) were untreated or stimulated with IL-17A (50ng/ml) for 0, 2, 6 or 24 hours, as indicated. The mRNA and proteinlevels were then analyzed by RT-PCR (Top) and Elisa (Bottom),respectively. Data are representative of two independent experiments(mean and s.d., *, p<0.05, **, p<0.01 by Student's t-test).

FIGS. 5A-0 . Act1 forms distinct RNPs with Dcp1/Dcp2, SF2 and HuR. A.Confocal imaging of in situ PLA in HeLa cells transfected with theindicated expression constructs. Primary mouse antibodies againstFlag-tag and rabbit antibodies against HA-tag were used. The red dotsindicate the interaction of the two proteins including Act1-HA/SF2-Flag,Act1ΔSEF-HA/SF1-Flag, Act1-HA/HuR-Flag and Act1-ΔSEF-HA/HuR-Flag. Nucleistained with DAPI. The expression levels of the transfected constructswere also analyzed by western analyses shown below the imaging data. B.Bar graph shows the percentages of PLA positive cells shown in A. Thequantification is based on the analysis of 50 cells, followed bytwo-tailed Student's t-test. Data represent mean±SD; *, p<0.05. C.Act1−/−MEFs reconstituted by retroviral infection with eitherFLAG-tagged mouse wild-type Act1 (WT) or SEFIR1 deletion mutant werepre-treated with TNF for 1 hour and then treated with IL-17 for 0 and 60min followed by RNA immunoprecipitation with anti-SF2 or anti-HuR,followed by RT-PCR analyses of the indicated mRNAs. The presented arethe relative values normalized against IgG control (Material andMethods). D. Schematic representation of the mouse CXCL1 3′UTR. HuR,Act1 and SF2 binding regions are indicated. E. Binding of purifiedrecombinant SF2 to SEFIR Binding Element (SBE WT, CXCL1 47 as indicatedin A) and stem-loop B and stem-loop C disruption mutants (SBE mutant Band SBE mutant C, Table 2) were examined by REMSA. F. Act1-SEFIR and SF2RNA binding competition was performed using probe CXCL1 220 II(containing SF2 and Act1 binding site as indicated in D). RadiolabeledRNA probe was incubated with increasing amounts of Act1-SEFIR with orwithout co-incubation with purified SF2 as indicated. Free RNA wasseparated from RNA-protein complexes by native PAGE. Graph indicates thedissociation of SF2-CXCL1 220 II upon incubation with increasing amountsof Act1-SEFIR, which was calculated by quantifying the remainingSF2-CXCL1 220 II complex in the presence of indicated amounts ofAct1-SEFIR. Data are representative of three independent experiments(mean and s.d.). G. Cytoplasmic and nuclear fraction lysates fromAct1−/−MEFs reconstituted by retroviral infection with eitherFLAG-tagged mouse wild-type Act1 (WT) or SEFIR1 deletion mutant,stimulated with IL-17 (50 ng/ml) for the indicated times, wereimmunoprecipitated with anti-FLAG and analyzed by Western blots with theindicated antibodies. The experiments were repeated for two times. H. Invitro kinase assay of purified recombinant IKKi using purifiedrecombinant SF2 as a substrate. I. REMSA was performed to examine thebinding of IKKi-phosphorylated SF2 (p-SF2) and non-phosphorylated SF2(minus IKKi and minus ATP) to SEFIR Binding Element (SBE WT, CXCL1 47 asindicated in FIG. 3A). Graph indicates the apparent Kd of CXCL1 47 (FIG.3A), which was calculated as the concentration of SF2 protein requiredto achieve 50% binding of the RNA (Material and Methods). Data arerepresentative of three independent experiments (mean and s.d.). J.Binding of purified recombinant HuR to CXCL1 220-I (containing HuRbinding site) and CXCL1 220 II (containing SF2 and Act1 binding site) asindicated in D were examined by REMSA. K. The simultaneous binding ofpurified recombinant HuR and Act1-SEFIR to CXCL1 3′UTR was examined byREMSA using CXCL1 220 (containing HuR and Act1 binding site as indicatedin D) and CXCL1 220 II (containing Act1, but not HuR binding site asindicated in D) as probes. The co-binding Act1-HuR was observed withprobe CXCL1-220, but not CXCL-220 II. L. UV-absorbance profile of RNPand polysome complexes separated on a sucrose density gradient intodifferent fractions as indicated. M. Cytoplasmic extracts of Act1−/−MEFsreconstituted by retroviral infection with either FLAG-tagged mousewild-type Act1 (WT) or SEFIR1 deletion mutant, pre-treated with TNF for1 hour and then treated with IL-17 for 0 and 90 min, were fractionatedthrough a 10-50% sucrose gradient (as described in L) and analyzed byWestern blot analyses with the indicated antibodies. N. CXCL1 and GAPDHmRNAs from translation-active pools and translation-inactive pools fromL were analyzed by RT-PCR and normalized to β-actin. Graph shows theratios of mRNAs from translation-active/inactive pools. Data arerepresentative of three independent experiments. (mean and s.d., *,p<0.05, **, p<0.01 by Student's t-test). O. Model of Act1-RNP in mRNAstabilization: Act1 directly binds to the mRNAs of inflammatory genes toform multiple RNPs controlling different steps of mRNA metabolism inresponse to IL-17 stimulation. Upon IL-17 stimulation, Act1 istranslocated into the nucleus where Act1 binds to a stem-loop structurein the 3′UTR in the target mRNAs (RNP1). The binding of Act1 competesoff SF2 from the mRNAs by bringing IKKi to phosphorylate SF2, preventingSF2-mediated mRNA decay. Act1 then follows the mRNAs to the P-bodies(RNP2) inhibiting Dcp1/2-mediated mRNA decapping by employing TBK1 tophosphorylate Dcp1. Finally, Act1-mRNAs are shifted to the polysomes tofacilitate HuR's binding to mRNAs (RNP3) for protein translation.

FIGS. 6A-F. SBE RNA aptamers abolished IL-17-induced mRNA stabilizationof CXCL1, GM-CSF and TNF. A. SBE RNA aptamers were used to compete withCXCL1 220 probe for binding to Act1-SEFIR. The purified recombinantHis-MBP-Act1 SEFIR (200 ng) was incubated with the CXCL1 220 in thepresence of indicated folds of excess SBE aptamers followed by REMSA.SBE WT (CXCL1 47 as indicated in FIG. 3A), SBE mutant B and SBE mutant C(Table 2) were used in the competition assay. Graph quantifies theremaining Act1-SEFIR-bound CXCL1 220 probe in the presence of indicatedconcentrations of SBE aptamers. IC50 is calculated as the concentrationof aptamers that displace 50% of bound CXCL1 220 probe from theAct1-SEFIR. Data are representative of three independent experiments(mean and s.d.). B. Confocal imaging was used to analyze thelocalization of RFP-Act1 and fluorescein amidite labeled SBE aptamer(SBE-FAM). HeLa cells were transfected with Act1-RFP, followed by asecond transfection with SBE-FAM 24 hours later. Cells were fixed atindicated hours after SBE-FAM transfection and analyzed using confocalmicroscopy. Nuclei were stained with DAPI. Graph indicates the averagenumbers of SBE-FAM-positive, Act1-RFP-positive andSBE-FAM/Act1-RFP-double positive granules per cell at 5, 10 and 20 hoursafter SBE-FAM transfection. The quantification is based on the analysisof 50 expressing cells, followed by two-tailed Student's t-test. Datarepresent mean±SD; *, p<0.05, **, p<0.01. C. Confocal imaging ofDcp1-RFP and Act1-GFP expressing cells transfected with 100 pmoles/ml ofunlabeled SBE aptamers. HeLa cells were co-transfected with Dcp1-RFP andAct1-GFP, followed by a second transfection with unlabeled SBE aptamers24 hours later. Cells were fixed 24 hours after SBE aptamerstransfection and analyzed using confocal microscopy. Nuclei were stainedwith DAPI. Graph indicates the average numbers of Dcp1-RFP-positive,Act1-GFP-positive and Dcp1-RFP/Act1-GFP-double positive granules percell 24 hours after SBE-FAM transfection. The quantification is based onthe analysis of 50 expressing cells, followed by two-tailed Student'st-test. Data represent mean±SD; *, p<0.05. D. SBE RNA aptamers were usedto compete with GM-CSF 3′UTR (513-785) and TNF 3′UTR (1362-1507) probesfor binding to Act1-SEFIR. The purified recombinant His-MBP-Act1 SEFIR(200 ng) was incubated with the GM-CSF and TNF 3′UTR probes in thepresence of indicated folds of excess SBE aptamers followed by REMSA.SBE WT (CXCL1 47 as indicated in FIG. 3A) and SBE mutant C (Table 2)were used in the competition assay. E. RNA decay in HeLa cellstransfected with or without 100 pmoles/ml of SBE RNA aptamers (WT ormutant C), pre-treated with TNF (10 ng/ml) for 1 hour and then treatedwith Actinomycin D alone (NT) or in the presence of IL-17 (50 ng/ml) for45 and 90 minutes. Human CXCL1, GM-CSF and TNF mRNAs were measured byRT-PCR, normalized to GAPDH and presented as half-life. F. HeLa cellstransfected with or without 100 pmoles/ml of SBE RNA aptamers (WT ormutant C) were pre-treated with TNF (10 ng/ml) for 1 hour followed bystimulation with IL-17A (50 ng/ml) for 6, 8 or 24 hours. Supernatants ofthe treated cells were then analyzed by ELISA. Data are representativeof two independent experiments, and analyzed by two-tailed Student'st-test. Data represent mean±SD; *, p<0.05, **, p<0.01.

FIGS. 7A-D. SBE RNA aptamers inhibited IL-17-dependent skin hyperplasiaA. H&E staining of ear skin tissue from C57BL/6J mice injectedintradermally with IL-17A (500 ng) or PBS together with methylated(Materials and methods) SBE RNA aptamers [SBE mutant C (1 nmol) as anegative control or SBE WT aptamer (1 nmol)] for 6 consecutive days. B.Paraffin sections of the ear skin from A were subjected toimmunohistochecmitry staining for Ly6G. C. Epidermal thickness of earskin from A were measured in arbitrary units and the average number ofLy6G+ cells per section from B were counted. 5 fields were analyzed foreach mouse under 20× magnification. Scale bar is 100 μm. Data areanalyzed by two-tailed Student's t-test; *, p<0.05, t-test. Datarepresent mean±S.E.M of biological replicates (n=6 mice/group). D.RT-PCR analysis of mRNA isolated from the ear skin tissue as describedin A. Expression of Act1-target genes was graphed as mean fold inductionover PBS+ control aptamer treated group. Data represent mean±SEM ofbiological replicates (n=6 mice/per group) and. Two-tailed Student'st-test were used to analyze the data. **, p<0.01, *, p<0.05.

FIG. 8 shows a hypothetical, non-limiting, mechanism of ACT-1 mediatedmRNA stabilization affecting inflammatory gene expression inpathogenesis of autoimmune and inflammatory diseases, and therapeuticintervention with nucleic acid sequences that selectively binding ACT1with inhibitory activity against IL-17.

FIG. 9 shows a hypothetical, non-limiting, mechanism of usingaptamer-mediated Act1-mRNA disruption, to treat inflammatory andautoimmune diseases, such as psoriasis, rheumatoid arthritis, multiplesclerosis, inflammatory bowel diseases, and systemic lupus.

FIG. 10 shows an SBE from the mouse CXCL1 mRNA that is 2′-O-methylated(SEQ ID NO:80), including the step and loop secondary structure formedby this sequence.

FIG. 11 shows a consensus structure of an exemplary SBE nucleic acidaptamer (SEQ ID NO:1). The N's in these sequences are independentlyselected from A, G, C, T, U, as well as modified and non-canonicalnucleotide bases. As can be seen, if a particular N is chosen, the baseit is hybridized to must be a corresponding base (e.g., if “G” ischosen, the hybridized base will be “C”).

FIG. 12 shows a consensus structure of an exemplary SBE nucleic acidaptamer (SEQ ID NO:2). The N's in these sequences are independentlyselected from A, G, C, T, U, as well as modified and non-canonicalnucleotide bases.

FIG. 13 shows seven exemplary SBE nucleic acid aptamers (SEQ IDNOS:88-94).

FIG. 14 shows four exemplary SBE nucleic acid aptamers (SEQ IDNOS:99-102).

FIG. 15 shows a consensus structure of an exemplary SBE nucleic acidaptamer (SEQ ID NO:103), where the three “Ws” are either A or U (e.g.,AAA, AUA, UUA, UUU, UAU, etc.).

FIG. 16 shows eight exemplary SBE nucleic acid sequences (SEQ IDNos:104-111) that showed positive results when tested by EMSA in workconducted during development of embodiments of the present disclosure.

DETAILED DESCRIPTION

Provided herein are compositions, systems, kits, and methods fortreating IL-17a related diseases and conditions using an SBE nucleicacid sequence that binds a SEFIR domain of an ACT1 protein. In certainembodiments, the SBE nucleic acid sequences (aptamers) comprise RNAand/or DNA, and are between 10 and 65 nucleotides in length. In certainembodiments, the SBE aptamer forms a stem and loop type structure.

Proinflammatory cytokine IL-17, a major driver of autoimmunity, signalsthrough a heterodimeric receptor complex (IL-17RA and IL-17RC), whichinteracts with the SEFIR-containing adaptor, Act1. Work conducted duringthe development of embodiments descried herein found that Act1 has anon-canonical function as an RNA binding protein that stabilizesotherwise unstable mRNAs of the pro-inflammatory genes in response toIL-17 stimulation including CXCL1, TNF, GM-CSF, as well as IL-6.Structure-functional analysis showed that the SEFIR domain is necessaryand sufficient for the RNA binding activity of Act1. While the presentinvention is not limited to any particular mechanism, and anunderstanding of the mechanism is not necessary to practice theinvention, it is believed that Act1 directly binds to a stem-loopstructure in the 3′UTR of the mRNAs in P-bodies, inhibiting theirdecapping by bringing a kinase, TBK1 to phosphorylate and disruptDcp1/Dcp2 complex. RNA oligos containing the Act1 RNA binding motifabolished Act1's localization in P-bodies and prevented Act1-mediatedmRNA stabilization. Taken together, these results support a new paradigmin which the receptor-proximal adaptor Act1 directly controls mRNAmetabolism, providing a mechanism for receptor-mediated selectivity ofmRNA stabilization during inflammation.

Work conduced herein has shown that Act1, an interleukin-17 (IL-17)receptor complex adaptor, binds and stabilizes mRNAs encoding keyinflammmatory proteins. The Act1 SEFIR domain binds a stem-loopstructure, SBE (SEFIR-binding element), in the CXCL1 3′UTR. Remarkably,mRNA-bound Act1 directs formation of three compartmentally-distinct RNPsthat regulate three disparate events in inflammatory mRNA metabolism,preventing SF2-mediated mRNA decay in the nucleus, inhibitingDcp1/2-mediated mRNA decapping in P-bodies, and facilitating HuR bindingto polysomal mRNA to promote translation. SBE RNA aptamers reducedIL-17-mediated mRNA stabilization in vitro, and IL-17-induced skininflammation, providing a new therapeutic strategy for autoimmunediseases and related conditions. While the present invention is notlimited to any particular mechanism, and an understanding of themechanism is not necessary to practice the invention, it is believed,taken together, these results reveal an extraordinary network in whichAct1 assembles RNPs on the 3′UTRs of select mRNAs to controlreceptor-mediated mRNA stabilization and translation duringinflammation.

In certain embodiments, provided herein are SBE aptamers, which may beused to treat IL-17a disease or conditions. In certain embodiments, theSBE aptamer is a single stranded RNA or DNA oligonucleotide (e.g.,12mer-70mer, such as a 56-mer) that binds to ACT1 and inhibits theactivity of IL-17a. In certain embodiments, the SBE aptamers are,compared to antibodies, are smaller in size, lipophilic and enter cellsmore easily, and have lower immunogenicity. In particular embodiments,the SBE aptamers inhibit IL-17 mediated mRNA stabilization. Inparticular embodiments, the SBE aptamers recognize ACT-1 (throughbinding) with specificity. In certain embodiments, the SBE aptamersinhibit IL-17 in vivo without losing the anti-bacterial and anti-fungalfunction of IL-17. In certain embodiments, the SBE aptamers are furtherchemically modified to improve affinity and/or increase stability (e.g.,2′-O-methylation). In some embodiments, the SBE aptamers, at one or morepyrimidine nucleotides, modifications are made at the 2′-sugar or basefor improved affinity and/or stability.

IL-17a is a known target in the pathogenesis of many inflammatory andautoimmune diseases. COSENTYX (secukinumab) is a selective binding toIL-17 which is approved in the EU for the treatment of Psoriasis. TheSBE aptamers herein may be used as alternative therapy to secukinumab totreat Psoriasis or other autoimmune diseases.

In certain embodiments, the SBE nucleic acid aptamer sequence isselected from SEQ ID NOS:2 and 88-94, which are shown in FIGS. 12 and 13, and in Table 4 below.

TABLE 4 N1GN2UAAUAN3CN4 (N1/N4, N2/N3 = G/C or A/U) SEQ ID NO: 2GUUGGUAGGGCAUAAUGCCCUUUU SEQ ID NO: 88CCUUGGACAUUUUGUGUCUAGUUGGUAGGGCAUAAUGCCCUUUUACA SEQ ID NO: 89UUUGUGUCUAGUUGGUACCCCAUAAUGGGGUUUUACAUUCUUUA SEQ ID NO: 90UUUUCUCUCUAGUUGGUAAGGCAUAAUGCCAUUUUAC SEQ ID NO: 91UUGCCUCCUCUUUUUUAUGCUUAAAGCAAAAUAUUUA SEQ ID NO: 92UUUAUUGAUAAUUUAUAUAAAUAACCUUAAAGGUAAAAUAUGAUUGAU SEQ ID NO: 93CCUUGGACAUUUUGUAUAUAGUUGGUAGGGCAUAAUGCCCUUUUACA SEQ ID NO: 94

In certain embodiments, the SBE nucleic acid aptamer sequence isselected from SEQ ID NOS:112-129, which are shown in Table 7 below(where N is selected from A, T, G, C, and U).

TABLE 7 NNNNNNNNNNNNAGAUAAUAUCUNNNNNNNNNNNNN SEQ ID NO: 112NNNNNNNNNNNNCGAUAAUAUCGNNNNNNNNNNNNN SEQ ID NO: 113NNNNNNNNNNNNAGCUAAUGUCUNNNNNNNNNNNNN SEQ ID NO: 114NNNNNNNNNNNNCGCUAAUAGCGNNNNNNNNNNNNN SEQ ID NO: 115NNNNNNNNNNNNUGAUAAUAUCANNNNNNNNNNNNN SEQ ID NO: 116NNNNNNNNNNNNUGUUAAUAACANNNNNNNNNNNNN SEQ ID NO: 117NNNNNNNNNNNNGGAUAAUAUCCNNNNNNNNNNNNN SEQ ID NO: 118NNNNNNNNNNNNAGGUAAUACCUNNNNNNNNNNNNN SEQ ID NO: 119NNNNNNNNNNNNGGGUAAUACCCNNNNNNNNNNNNN SEQ ID NO: 120NNNNNNNNNNNNAGUUAAUAACUNNNNNNNNNNNNN SEQ ID NO: 121NNNNNNNNNNNAAGAAAAUCUUNNNNNNNNN SEQ ID NO: 122NNNNNNNNNNNAAGAAUAUCUUNNNNNNNNN SEQ ID NO: 123NNNNNNNNNNNAAGAAAUUCUUNNNNNNNNN SEQ ID NO: 124NNNNNNNNNNNAAGAAUUUCUUNNNNNNNNN SEQ ID NO: 125NNNNNNNNNNNAAGAUAAUCUUNNNNNNNNN SEQ ID NO: 126NNNNNNNNNNNAAGAUAUUCUUNNNNNNNNN SEQ ID NO: 127NNNNNNNNNNNAAGAUUAUCUUNNNNNNNNN SEQ ID NO: 128NNNNNNNNNNNAAGAUUUUCUUNNNNNNNNN SEQ ID NO: 129

In certain embodiments, one, two, or three nucleotide substitutions aremade to SEQ ID NOS:88-94 and 99-129, such as a conservative substitutionthat does not substantially change the stem and loop structures shown inFIG. 13 or 14 . In other embodiments, one, two, or three nucleotides aredeleted from the 5′ end, the 3′ end, or both the 5′ and 3′ ends of SEQID NOS:88-94 and 99-129 to generate truncated versions thereof.

In some embodiments, the additional SBE nucleic acid sequence aptamersthat bind ACT1 SEFIR domain can be identified. Provided in Table 6 belowis 644 transcripts that are stabilized by IL-17 stimulation in culturedkeratinocytes. These transcripts, as well as mutated versions of SEQ IDNOS:88-94 and 99-129, could be screened for SBE sequences that bind ACT1SEFIR domain. For example, one identify SBE aptamers from the 3′UTRs ofthe transcripts in the gene list in Table 6 by measuring the binding ofan RNA aptamer containing sequence derived from the aforementioned 3′UTRs to purified Act1 SEFIR protein (SEQ ID NO:95,rkvfitysmdtamevvkfvnfllvngfqtaidifedrirgidiikwmerylrdktvmfivaispkykqdvegaesqldedehglhtkyihrmmqiefisqgsnmfrfipvlfpnakkehvptwlqnthvyswpknkknillrllree) using electrophoretic mobility shift assay(EMSA), surface plasmon resonance assay or microscale thermophresisassay (see Example 1 further below for conditions and procedures).Candidate RNA aptamers that bind to Act1-SEFIR protein are consideredSBE aptamers, which have the potential of ameliorating IL-17 mediateddiseases.

The identified SBE can be further tested for their ability to inhibitIL-17 induced mRNA stability. To this end HeLa cells could betransfected with or without 100 pmoles/ml of SBE RNA aptamers,pre-treated with TNF (10 ng/ml) for 1 hour and then treated withActinomycin D alone (NT) or in the presence of IL-17 (50 ng/ml) for 45and 90 minutes. Human CXCL1, GM-CSF and TNF mRNAs could be measured byRT-PCR, normalized to GAPDH and presented as half-life. Supernatants ofthe treated cells are analyzed by ELISA. SBE aptamers that can reducethe half-life of CXCL1, GM-CSF, and TNF transcripts and blunt theirprotein expression are bona fide SBE aptamers that can be used to treatIL-17 mediated diseases such as psoriasis, severe asthma and cancer.

TABLE 5Example SBE element identified from 3’UTRs of transcripts in Table 6CTTTTGCTTATGTTTAAAACAAAAT (SEQ ID NO: 96) TNF derived SBEAGTAAACTTTAAGTTAATTTAUG (SEQ ID NO: 97) GMCSF derived SBECTGACCCTGATACAGGCATGGCA (SEQ ID NO: 98) IL-6 derived SBE

TABLE 6 GENE NAME Cxcl1 II13ra2 Gzmf Klrb1f Trim50 Fgd5 Tnf Arl9 Tmem125Stoml3 Tmem52 Gsdmcl1 Csf2 Opn1mw Nobox Apeg3 Atp6v1b1 H2-Q7 Il6 Dppa3Zg16 Igll1 Rab33a Stmn3 Saa3 Hs3st6 Fcamr Ifnk Bpifa2e Tcfap2c CampII17c Sprr2k Efcab3 Glp2r Pnma5 Serpina3i Lect2 Ccl24 Gtsf1I Olfr1450Tmem145 Mir16-2 AI428936 Niacr1 Smtnl1 Cacng4 Cdr1 Mir345 Atp6v1c2Clec2f-ps Mospd4 Nos2 Gpr87 Hist1h2ad Cd300c Lce1I Cplx4 Spata21 CideaMir350 Olfr1420 C2cd4b Ctf2 H2-Q8 H2-BI Hils1 Gzmc Dleu7 Syce1I AvpTmem196 Mir674 CapsI Cyp1a1 Prl8a8 Pxt1 Alms1-ps2 Mir1970 Dnajc5b TrehVmn1r38 Tceal5 Olfr1182 Mir297b Gngt1 Ltb4r1 Igfbpl1 Lhb Mmel1 Mir449cGpx2-ps1 Ascl3 Nepn H2-M9 Serpina7 Mir199b Apoc1 C1ql4 Olfr992 Cxcl3Xlr4a Rfpl3s Krt23 Tmem163 Faim2 Tcl1b4 Gstm3 Olfr1314 Msmb Iapp Prl8a9Tssk3 Ssxb5 Bglap2 Mmp21 Cd300lh Tas2r140 Ms4a10 Cpne7 Scarna8 Pkp3Vgll2 Olfr577 Hrasls5 Cldn27 Lcn2 Gpr84 Fbxw13 Spaca3 Trem3 Iqub Mir1192Taar7d Plcd4 Olfr1278 Olfr421 Slc6a19 Mir669h Tcte3 Lipn Olfr457 Nctc1Lypd6 Snora62 Olfr419 Ppef2 Morn3 Slc4a9 MarveId3 Otor BC024582 Unc93aTnip3 St8sia5 Slc25a41 Vmn1r40 Csn2 C4a Olfr429 Tmprss2 Trpc3 Gng13Serpina3f Helt Atp6v1g3 Nkx2-4 Btnl9 Cd83 Tpte Slc5a11 Olfr1474 Olfr374Myh7b Cited4 Wisp3 Cacng6 Olfr1475 Sox7 Zfhx2as Kcnk15 Oaz3 Tuba3b Cma2Olfr172 Fcgr4 Morn5 Darc Slc2a7 Slc6a20a Kcng4 Trim63 Colec11 Otud6aPrss42 Vmn1r205 Rlbp1 Foxl2 Ly6d Cxcl5 Slc15a2 Olfr1418 A4gnt Olfr225Art1 Krt8 Tdrd12 Olfr572 Spata3 Gpx6 Tmem212 Npw Gp9 Fmr1nb Speer1-ps1Rnf186 Ppbp Mx2 Cyp2d26 Olfr1347 Pate2 Spata22 Trp53tg5 Gabrr2 Ribc2Xkr9 Elf5 Tekt5 Catsper3 Gpr97 Myo1g Serpinb9c Nsg2 Fcrlb Rpp25 Prrt1Pnpla3 Olfr726 Hs3st1 Prss40 II12a Apobec4 Fam151a Wnt9b Hes5 Wnt3Fam50b Dpf3 Cct8l1 Zfp352 Srms Fstl4 C2cd4d Zdhhc22 Olfml1 Apol11aVmn2r21 Oca2 Hrh3 Hsd3b5 Hoxd11 Kif2b Vmn2r15 Sox17 Itgad Amph InhbcPabpn1I Pabpc2 Ptpn5 Ccdc54 Pnliprp1 Sigirr Dgki Vmn2r38 lgdcc3 II1bPrb1 Mmp1b BB123696 Ly6h Ntn5 Clec4a3 Kbtbd12 Prss55 Slc27a2 Pcdha7Prdm13 Nat1 Lrrc3b Zp3r Asb10 Pcdha6 Samd7 Serpina4-ps1 Kcng3 H2-Q1Wfdc5 Ppp1r1c II7r Fam123a Fbxo40 Tex19.2 Barhl2 Slfn4 Hoxa9 Rpl3lMcoln3 Prf1 Sec14l5 Scnn1b Foxo6 Gdf3 Plcb2 Hapln2 Spock1 Gpr128 Lst1II27 Steap4 II4i1 Capn9 Dmbx1 Ptchd3 Abp1 Hsd17b13 Cyp3a41b Adam30 Cwh43Ankmy1 Lta Bpifb2 Fam115e Fam43b Csprs Nefm Batf2 Lim2 Mrap2 Slc47a2Tyrp1 Ffar3 Tlx2 Epb4.2 Kcne1 Ccl20 Adam29 Cd101 Fam19a4 Treml4 Plk5Cldn10 Zfp957 Klhl4 Prss32 Slc22a3 Dyrk4 Mageb4 D6Ertd474e Gabrp AanatTacstd2 Vax2os1 Pou6f2 Slc35f3 Ccdc129 H2-Q10 P2ry4 II25 Clcnka Slco1a6Clec9a Cldn14 Oxct2a Tgm7 Syt1 Nell1 Frem3 Atp1b2 Itpka Asb15 Gpr132Pglyrp2 Car10 Zic2 Dlx4 Bpifb9a Rbpjl Tnfrsf9 BC147527 Fam187b Klk14Snap25 Krt1 Cacng2 Nlrp4e Klf14 Ggt6 Fam19a3 Speer4f Serpinc1 Syt2Grin2a Uox Serpina5 Clec2g Aurkc Ccdc146 Arl11 Rit2 Hpse2 II2ra2 Ubxn10Ms4a15 Tssk5 Alx3 Tbc1d21 Prss38 Cckar Krt78 Camkv Epsti1 H2-Q2 Capn8Fam163b Oasl2 Wnt8a Padi1 Zcchc12 Tmem90a Foxb1 II1a Fbxw15 Olfr1310Lhx3 Nrl Ccbp2 Neurl3 Chrdl2 Cd244 Slc22a29 Acsm5 Accn1 Tchh Igfbp1Timm8a2 Dspp Dnaic1 Kcnk18 Odf4 Tph1 Crtam Nlrp6 Hes2 Fbxw16 Tlr9 Tlx3Plcxd3 Tnfaip8l3 Pou2f3 Fer1l4 Inpp5j Slco2b1 Cyp8b1 Olfr1300-ps1 Tgm5Lax1 Lrrn4 Wt1 Kif17 G6pc Kcnh7 Htr7 Emr4 Lrrc66 Tm4sf5 Trim31 Lrrtm1Ccdc33 Xpnpep2 Ybx2 Slc36a2 Crxos1 Dpysl5 Kcng11 Kcnk4 Tdrd6 Acvr1cNlrp3 E330023G01 lkzf3 Vmn2r28 Ermn Dlgap2 Otx1 Bcl3 Zfp804a Slc39a12Naip1 Ccnb3 Rd3 Lce1g Igj Hlf Cdh22 Scn4a Nppc Clstn3 Gja6 Dydc2Serpina3h ligp1 Vipr1 Spem1 Lhfpl3 Kbtbd5 Spag16 Ccr5 Nlrp2 II1rapl1Tmem132d Cp Pcnxl2 Masp2 Slc22a2 Cryba1 Krt73 Ccl7 Prodh2 Ctrl Plg Dub1Pnoc Zc3h12a Dnase1l3 Ncam2 Brs3 H28 Dll3 Fzd10 Nr5a2 Hrc Sv2b CgnNkx2-2as Zmynd10 Hcrtr2 Dnahc2 Gck Tdrd9 Rny3 Fscn3 Eomes Gria1 Creg2Ppp1r3a Ehf Scn10a C3 Sucnr1 Kcnn3 Glra4 Best2 Hhatl Dsc1 Eps8l3 Ttc9bProkr2 Dmrt1 Atp2c2 Fn3k Vwc2l Serpina3g Cidec Tacr2 Myo5c Olfr536 Padi6AU022754 Mettl7b Sall4 Zswim5 Rasd2 Cps1 Rny1 Hc Cd86 Robo3 B4galnt4Crym Cacna1f Nhlrc4 Ifi203 Ccdc147 Cpne4 Nfkbiz Myh6 H60b Fabp12 Kcnk10Clca4 Pak7 Ugt2b34 Rimklb Vmn2r24 Mmp25 Tcp11 Fstl5 Ccdc63 BC030870 Rbp3Acap1 Cacna1e Glb1l3 Slamf8 Ttc34 Gys2 Ccdc67 Piwil1 Slc17a4 Hoxa10 C8gLrat Fam24a Catsperg2 Vmn2r2 Atp7b Rab44 Pgr15l Papln Ccl2 Tmem229aGabbr2 Atp1a4 Pcdha2 Kng1 Arhgap40 Zf12 Psapl1 Clvs2 C87414 Fxyd2Apol10b

EXAMPLES Example 1

In this Example, an exciting and novel role of Act1 is reported, whichfunctions as a direct RNA binding protein to stabilize otherwiseunstable mRNAs of pro-inflammatory genes in response to IL-17stimulation, including CXCL1, TNF and GM-CSF. Mutagenesis studiesindicate that Act1 directly binds to a stem-loop structure in the 3′ UTRof CXCL1, and the SEFIR domain in Act1 is necessary and sufficient forthe RNA binding activity. In support of this, exemplary RNA aptamerscontaining the stem-loop structure (referred as SBE), inhibited Act1'sbinding to the target mRNAs and attenuated IL-17-mediated mRNAstabilization. Moreover, while SBE RNA aptamers inhibited IL-17-inducedskin inflammation.

While the present invention is not limited to any particular mechanism,and an understanding of the mechanism is not necessary to practice theinvention, it is believed that mechanistically, Act1 directly binds tothe mRNAs of inflammatory genes to form multiple RNPs (protein-RNAcomplexes) controlling different steps of mRNA metabolism. Act1 binds tothe mRNAs in the nucleus (RNP1) preventing SF2-mdiated mRNA decay bycompeting off SF2's binding to mRNAs, and Act1 follows the mRNAs to theP-bodies (RNP2) inhibiting Dcp1/2-mediated mRNA decapping by employingTBK1 to phosphorylate Dcp1. Finally, Act1-bound mRNAs are shifted to thepolysomes by facilitating HuR's binding to mRNAs (RNP3) for proteintranslation. Taken together, this Example provides the first example ofa receptor-interacting adaptor molecule, Act1, playing a direct role inmRNA metabolism, and elucidates a new mechanism for receptor-mediatedselectivity of mRNA stabilization and translation.

Materials and Methods:

Animals: IKKi-deficient mice were a gift from T. Maniatis. TBK1 floxmice were obtained from Millenium Pharmaceuticals, Inc., Mice 6 weeks ofage were used for primary kidney cells isolation. LSL-HA-Act1 knock-inmice were bred onto UBC-Cre-ERT2 mice (Ruzankina et al., 2007). TheCleveland Clinic Institutional Animal Care and Use Committee reviewedand approved the animal experiments.

Cell culture and Reagents: Antibodies against Act1, GAPDH and β-actinwere from Santa Cruz Biotechnology. Anti-hemagglutinin (HA) was fromSigma; TBK1 antibody, M2, V5, p-JNK, JNK, p-IkBa, p-p65, p65, IKKi,IKKa/β, pERK and pIkB antibodies were purchased from Cell SignalingTechnology. p-5315 Dcp1a antibody was a kind gift from Dr. ElisaIzaurralde. Dcp1 antibody was a kind gift from Dr. Jens-Lykke Andersen.TBK1 inhibitor MRT67307 was purchased from Sigma Aldrich. Cell cultureof mouse embryonic fibroblasts (MEFs), HeLa Tet-Off cells and primarykidney epithelial cells were isolated as previously described (Herjan etal., 2013). Act1−/−MEFs were reconstituted with either empty vector,flag-tagged mAct1, or flag-tagged deletion mutants of mAct1 byretroviral infection as described before (Liu et al., 2011).Proximity-based ligation assays were performed in Hela cells accordingto the manufacturer's instructions (Duolink™ Assay, Sigma Aldrich).

Transfection, adenoviral and retroviral infection: All transfectionswere conducted with Lipofectamine 2000 (Invitrogen) according to themanufacturer's instructions. For Act1 reconstitution into Act1−/−MEFs,cells were infected by retroviral supernatant as described previously(Qian et al., 2007). Briefly, viral supernatant was obtained bytransfecting Phoenix cells with 5 μg of retroviral construct derivedfrom pMSCV-IRES-GFP for 48 hours. MEFs were infected with viralsupernatant for 24 hours and GFP-positive cells were sorted out toestablish stable cell lines for downstream assay. Adenoviral infection:primary kidney epithelial cells were divided into 60-mm dishes andinfected by exposing them to media containing 2×10⁵ infectiousunits/plaque formation units of adenovirus/ml overnight.

Quantitative real-time PCR: Total RNA was isolated with TRIzol reagent(Invitrogen). The cDNA was synthesized with random hexamers (AppliedBiosystems) and M-MLV reverse transcriptase (Promega). Real-time PCR wasperformed using a SYBR Green PCR Master Mix kit (Applied Biosystems).All gene expression results were calculated by thechange-in-cycling-threshold (ΔCT) method, where ΔCT=CT of target gene−CTof Actb (encoding β-actin), and are presented as 2-ΔCT. The primers usedfor qPCR are listed in Table 3.

Constructs: For GFP reporter constructs, full-length cDNAs of hAct1,hDcp1a, hIKKi, hTBK1 and hTIA1 were cloned into pEGFP-N1 vector(Clontech). For RFP reporter constructs, full-length cDNAs of hAct1,hIRAK1, and hDcp1a were cloned into pDsRed-Monomer-Hyg-N1 vector(Clontech). For constructs V5-hDcp1a, FLAG-hDcp1a, FLAG-hDcp2,FLAG-hIRAK1, V5-IL-17RA, HA-hAct1 and HA-hAct1 ΔSEFIR, the cDNAs withthe corresponding tag were cloned into pcDNA3.1 vector. Wild-type(FLAG-hAct1) and Act1 deletion mutants (ΔSEFIR1, deletion of amino acidresidues 391 to 420; ΔSEFIR, deletion of amino acid residues 391 to 537)were flag-tagged and cloned into pcDNA3.1.

Constructs for in vitro transcription: Fragments containing the 3′UTRsequences of CXCL1 220 (nt 720-940) and the truncated fragments CXCL1120 (nt 780-900), CXCL1 110 (nt 780-890), CXCL1 90 (nt 790-880), CXCL180 (nt 800-880), CXCL1 70 (nt 800-870); SBE WT (CXCL1 47, nt 810-857),SBE mutant B (CXCL1 47, nt 810-857 with altered sequence shown in Table2), SBE mutant C (CXCL1 47, nt 810-857 with altered sequence shown inTable 2), Stem-loop B (CXCL1, 800-835) and Stem-loop C (CXCL1, 830-856)and as well as 3′UTR sequences of TNF (nt 1361-1507), GM-CSF (nt513-785) and GPx1 (nt 775-962) were generated by PCR and cloned intopGEM-3ZF(+) vector (Promega) using EcoRI and BamHI sites. The plasmidcontaining 3′UTR of mouse TNF were kind gift of Dr. Vigo Heissmeyer. Allmutations were introduced using QuikChange II Site Directed MutagenesisKit (Stratagene) according to the manufacturer's instructions. Primersused for generating all constructs are listed in Table 1.

TABLE 1 Primers used for PCR SEQ ID NO: CXCL1 220F: GGAAGAATTCCTGTGTTTGTATGTCTTG  3 R: GGAAGGATCCCTTTTATTTTTACTTCATTT  4CXCL1 120 F: GGAAGAATTCGTATGGTCAACACGCACGTGT  5R: GGAAGGATCCCTCTGTCCCGAGCGAGACG  6 CXCL1 110F: GGAAGAATTCGTATGGTCAACACGCACGTG  7 R: GGAAGGATCCCTCTGTCCCGAGCGAGACGAG 8 CXCL1 90 F: GGAAGGATCCCTCTGTCCCGAGCGAGACGAG  9R: GGAAGGATCCCTCTGTCCCGAGCGAGACGAG 10 CXCL1 80F: GGAAGAATTCTTGACGCTTCCCTTGGAC 11 R: GGAAGGATCCGACCAGGAGAAACAGGGTT 12CXCL1 70 F: GGAAGGATCCGACCAGGAGAAACAGGGTT 13R: GGAAGGATCCACAGGGTTAAAGAATGTAAAAGGG 14 CXCL1 47F: GGAAGAATTCCCTTGGACATTTTGTGTC 15 R: GGAAGGATCCTGTAAAAGGGCATTATGCC 16GPX1 F: CTGGTATCTGGGCTTGGTGATGG 17 R: CTGGTATCTGGGCTTGGTGATGG 18 TNF 150F: GGAAGGATCCTGTAAAAGGGCATTATGCC 19 R: GGAAGGATCCGCTTATGTTTAAAACAAAATAT20 GM-CSF F: GGAAGAATTCGACCGGCCAGATGAGGCTGGCC 21R: GGAAGGATCCCTTGAATAAATATGGAATATG 22 hAct1-GFPF: GGAAGCTAGCATGCCTCCTCAGCTTCAAG 23 R: GGAAGTCGACTGCAAGGGAACCACCTGAAG 24hDcpIa- F: GGAAGAATTCATGGAGGCGCTGAGTCGAGC 25 GFPR: GGAAGTCGACTGTAGGTTGTGGTTGTCTTTGTTC 26 hIKKi-GFPF: GGAAGCTAGCATGCAGAGCACAGCCAATTAC 27 R: GGAAGTCGACTGGACATCAGGAGGTGCTGGG28 hTBK1-GFP F: GGAAGGTACCATGCAGAGCACTTCTAATC 29R: GGAAGTCGACTGAAGACAGTCAACGTTGCG 30 hTIA-1-GFPF: GGAAGCTAGCATGGAGGACGAGATGCCC 31 R: GGAAGTCGACTGCTGGGTTTCATACCCTGCCAC32 hAct1-RFP F: GGAAGCTAGCATGCCTCCTCAGCTTCAAG 33R: GGAAGTCGACTGCAAGGGAACCACCTGAAG 34 hlRAK1-F: GGAAGCTAGCATGGCCGGGGGGCCGGGCCCG 35 RFPR: GGAAGTCGACTGGCTCTGAAATTCATCACTTTC 36 hDcp1a-F: GGAACTCGAGATGGAGGCGCTGAGTCGAGC 37 RFPR: GGAAGTCGACTGTAGGTTGTGGTTGTCTTTGTTC 38 V5-Dcp1aF: GGAAGCTAGCGCCACCATGGATTACAAGGATGACG 39 ATGACAAGATGGAGGCGCTGAGTCGAGCR: GGAAGAATTCTCATAGGTTGTGGTTGTC 40 FLAG-F: GGAAGCTAGCGCCACCATGGATTACAAGGATGACG 41 hDcp1aATGACAAGATGGAGGCGCTGAGTCGAGC R:GGAAGAATTCTCATAGGTTGTGGTTGTC 42 FLAG-F: GGAAGCTAGCGCCACCATGGATTACAAGGATGACG 43 hDcp2ATGACAAGATGGAGACCAAACGGGTGGAG R: GGAAGGATCCTCAAAGGTCCAAGATTTTC 44 FLAG-F: GGAAGCTAGCGCCACCATGGATTACAAGGATGACG 45 hlRAK1ATGACAAGATGGCCGGGGGGCCGGGCCCG R: GGAAGAATTCTCAGCTCTGAAATTCATCACT 46HA-hAct1 F: GGAAGCTAGCATGCCTCCTCAGCTTCAAG 47R: GGAAGAATTCTCACAAGGGAACCACCTGAAG 48

TABLE 2 Selected REMSA probes CXCL 220CUGUGUUUGUAUGUCUUGAAAAGAAUGUCAGUUAUUUAUUGAAAGUCGUCUUUCAUAUUGUAUGGUCAACACGCACGUGUUGACGCUUCCCUUGGACAUUUUGUGUCUAGUUGGUAGGGCAUAAUGCCCUUUUACAUUCUUUAACCCUGUUUCUCCUGGUCUCGUCUCGCUCGGGACAGAGACGUUCAAAGGACUGUUACAAAUGAAGUAAAAAUAAAAG (SEQ ID NO: 49) CXCL120GUAUGGUCAACACGCACGUGUUGACGCUUCCCUUGGACAUUUUGUGUCUAGUUGGUAGGGCAUAAUGCCCUUUUACAUUCUUUAACCCUGUUUCUCCUGGUCUCGUCUCGCUCGGGACAGAG(SEQ ID NO: 50) CXCL110GUAUGGUCAACACGCACGUGUUGACGCUUCCCUUGGACAUUUUGUGUCUAGUUGGUAGGGCAUAAUGCCCUUUUACAUUCUUUAACCCUGUUUCUCCUGGUCUCGUCUCGCU (SEQ ID NO: 51)CXCL90 GUAUGGUCAACACGCACGUGUUGACGCUUCCCUUGGACAUUUUGUGUCUAGUUGGUAGGGCAUAAUGCCCUUUUACAUUCUUUAACCCUGUUUCUCCUGGUCUCGUCUCGCU (SEQ ID NO: 52)CXCL1 80 UUGACGCUUCCCUUGGACAUUUUGUGUCUAGUUGGUAGGGCAUAAUGCCCUUUUACAUUCUUUAACCCUGUUUCUCCUGGUC (SEQ ID NO: 53) CXCL1 70UUGACGCUUCCCUUGGACAUUUUGUGUCUAGUUGGUAGGGCAUAAUGCCCUUUUACAUUCUUUAACCCUGUUUCUCCUGGUC (SEQ ID NO: 54) CXCL1 47CCUUGGACAUUUUGUGUCUAGUUGGUAGGGCAUAAUGCCCUUUUACA (SEQ ID NO: 55) (SBE WU)CXCL1 47 CCUUGGACAUUUUGUGUCUAGUUGGUAGGGCAUAAUGCCCUUUUACA (SEQ ID NO: 56)(SBE muUanUB) CXCL1 47CCUUGGACAUUUUGUGUCUAGUUGGUAAAACAUAAUGCCCUUUUACA (SEQ ID NO: 57)(SBE muUanUC) CXCL1 47CCUUGGACAUUUUGUGUCUAGUUGGUAGGGCAAGUUGCCCUUUUACA (SEQ ID NO: 58)(SBE AGU) CXCL1 47CCUUGGACAUUUUGUGUCUAGUUGGUACCCCAUAAUGGGGUUUUACA (SEQ ID NO: 59)(SBE GGG- CCC swap) CXCL1UUGACGCUUCCCUUGGACAUUUUGUGUCUAGUUGGU (SEQ ID NO: 60) (SUem loop B) CXCL1GUUGGUAGGGCAUAAUGCCCUUUUAC (SEQ ID NO: 61) (SUem loop C)

In vitro transcription and cap-labeling: REMSA radiolabeled 3′ UTR RNAprobes were synthesized from BamHI linearized plasmids (see constructsfor in vitro transcription) templates with T7 RNA polymerase using 1 mMGTP, 1 mM ATP, 1 mM CTP, 0.005 mM UTP and 25 μCi of 32P-labeled UTP for3 hours at 37° C. Probes were DNAse I treated for 20 minutes and thenphenol:chloroform extracted. The aqueous phase was passed through aMicro Bio-Spin P30 column according to manufacturer's instructions(BioRad). For in vitro decapping probes were synthetized as above, butusing un-labeled 1 mM CTP, DNAse treated and purified. Cap-labeling wasperformed using the vaccinia capping system (NEB) according to themanufacturer's instructions in the presence of [α-32P] GTP.

For RNase footprinting experiments, cold synthetic transcripts weredephosphorylated with SuperSAP (Affymetrix), purified, and resuspendedin nuclease-free water. Dephosphorylated transcripts were end-labeled inthe presence of [γ32P] ATP (3000 Ci/mmole; Perkin Elmer Easy Tides) andT4 PNK (NEB) at 20 units/pmole RNA. The transcripts were gel purified on8% acrylamide (19:1)/7 M urea gels and eluted in 10 mM Tris HCl, pH 7.5,1 mM EDTA, pH 8, 300 mM NaAc, pH 5.5 at 4° C. overnight. Purified RNAwas stored in 10 mM Tris HCl, pH 7.5 at −20° C.

RNase Footprinting

End-labeled 32P-labeled CXCL1 SBE RNA was heated to 95° C. and slowcooled to room temperature. The RNA (2.5 nM) was incubated in L30binding buffer (30 mM Tris HCl, pH 8.0, 75 mM KCl, 5 mM MgCl2, 1 mM DTT,0.04 μg/μL BSA (NEB), 10% glycerol, and 50 ng/μL yeast tRNA) with orwithout mouse Act1 SEFIR protein (1.5 μM) at 30° C. for 10 min.Reactions were cooled to room temperature over a 2 min period and thenplaced at 22° C. for 2-5 min. The indicated amounts of RNase T1, A, orV1 (Ambion) were added to the appropriate samples and incubated at 22°C. for 5 min. Enzymatic reactions were quenched with 30 μLInactivation/Precipitation buffer (Ambion) and purified according tomanufacturer's directions. Samples were resuspended in 10 μL of loadingbuffer (Ambion), heat-denatured at 95° C. for 5 min, and separated in adenaturing 8% (19:1) polyacrylamide/7 M urea gel. The dried gels werevisualized with a phosphorimager or on film.

Sequencing ladders were prepared by incubating end-labeled 32P-labeledCXCL1 SBE RNA (2.5 nM) in 1× Sequencing Buffer (Ambion) supplementedwith 50 ng/μL yeast tRNA. The RNAs were incubated at 50° C. for 5 min,cooled to 22° C. and the indicated amounts of RNase T1 and A added. Thesamples were incubated, quenched, and purified as described above.Alkali ladders were prepared by incubating end-labeled 32P-labeled PHGPxSECIS RNA (2.5 nM) in 100 mM NaOH, 2 mM EDTA, pH 8.0, and 2 μg/μL yeasttRNA at 37° C. for 3 min, to which 0.2 M Tris HCl, pH 8.0 (final) wasadded. The samples were frozen on dry ice and combined with an equalvolume of loading buffer.

RNA Electrophoretic Mobility Shift Assay (REMSA): Increasing amounts ofpurified protein and labeled probes (10 fmol, see in vitrotranscription) were combined in the binding buffer for 30 minutes. Thefinal REMSA binding buffer concentrations were 140 mM KCl, 10 mM HEPESpH 7.9, 5% glycerol, 1 mM DTT and 0.33 mg/ml tRNA. The reaction wasfurther supplemented with 15 μg salmon sperm DNA to reduce non-specificinteractions from the lysate. Complexes were resolved on either 4% or 6%non-denaturing polyacrylamide gels. The gels were dried and theappearance of complexes was visualized by exposure to BioMax MR film.Dissociation constants (Kd) were determined by quantified theprotein-bound fractions using ImageJ software and plotted againstprotein concentration (nM). Kd values were extracted from plots fittedto a hyperbolic function in Graph PAD Prism software (OriginLab).

Surface Plasmon Resonance: Binding affinity assays were conducted on aBiacore 3000. The biotinylated RNA was immobilized on astreptavidin-coated sensor chip. SA sensor chips was activated andblocked according to standard protocols. RNA was diluted to 1 mM inHBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005%surfactant P20) heated at 80° C. for 10 min, cooled to room temperatureto allow annealing of the stem, diluted 500-fold in running buffer. RNAwas injected at a flow rate of 10 μL/min 40 resonance units of RNA werecaptured on the SA chip. To study the RNA/Act1 interactions, theproteins were diluted in running buffer and injected at theconcentrations indicated in the sensorgrams. Binding experiments werecarried out at 25° C. and a flow rate of 30 μl/min. Any protein thatremained bound after a 3-min dissociation phase was removed by injecting2 M NaCl for 60 s at 20 μl/min, which regenerated the RNA surfacecompletely. Data were analyzed with BIAcore 3000 evaluation software andcurves were fitted with the “1:1 binding with drifting baseline” model.

Decapping assays: Dcp1/Dcp2 complex and Act1 protein were purified fromHeLa cells (2×106) either co-transfected with 10 ug of FLAG-tagged Dcp1and FLAG-tagged Dcp2 (kind gift from Dr. Andersen) or with 10 ug ofFLAG-tagged Act1 by using anti-FLAG/M2-beads (Sigma), eluted in 50 ulusing 3× FLAG peptide (Sigma) and protein concentrations were estimatedby comparison to a Act1 SEFIR protein of known concentration. 10 fmol of[32P] cap-labeled RNA substrate was incubated with the purifiedDcp1/Dcp2 complex (100 ng of each protein) and increasing amounts ofpurified Act1 protein (0, 100 and 300 ng) in decapping buffer (10 mMTris, pH 7.5, 100 mM KOAc, 2 mM MgOAc, 2 mM DTT) supplemented with fresh0.5 mM MnC12 for 30 min at 370 C. The reaction was terminated byaddition of 1 ul of 0.5 M EDTA. Reaction products were separated andidentified by TLC on cellulose sheets developed in 450 mM (NH4)2SO4.7meGMP and 7meGDP (20 μg) were spotted routinely on TLC plates alongwith reaction samples to serve as markers that could be visualized by UVshadowing.

Intradermal injection: LSL-HA-Act1 knock-in/UBC-Cre-ERT2 mice wereinjected with tamoxifen (˜5 mg/25 g weight) 14 and 7 days prior toIntradermal experiment. The ears of 8-week-old female mice were injectedintradermally with 20 ul of PBS alone or with PBS containing 0.5 mg ofrecombinant mouse IL-17A in the presence or absence of 5 nmol of eitherSBE WT aptamer or SBE mutant C aptamer (for sequence see Table 2). Micewere injected daily for 6 consecutive days. Six days after injection,skin tissue was collected for RNA and staining analyses. For H&E and DAB(3,3′-diaminobenzidine, BD phamagen) staining, skin tissue was fixed in10% formalin and then processed into paraffin blocks. Epidermalthickness was quantified by ImageJ software.

Aptamer design: Aptamers containing sequences of SBE WT and SBE mutant C(see Table 2) were ordered form Integrated DNA Technologies. Three firstresidues form both 5′ and 3′ end were methylated (2′-O-Methyl RNA bases)in order to enhance stability. For detection, aptamers were furthermodified at 5′ end with either 6-FAM or Atto647 fluorescent dyes.

Histological analysis: Tracheas were collected from LSL-Act1-HA KICre-ERT2 mice subjected to intranasal administration offluorescently-labeled SBE RNA aptamers, snap-frozen in OCT medium andcryosectioned at 5 μm. Frozen tissue sections was fixed andpermeabilized with 4% paraformaldehyde solution containing 0.2% TritonX-100 for 10 minutes. Sections were incubated with rabbit anti-HA Abfollowed by staining with Alexa Fluor 488-conjugated goat anti-mouse Ab(green) and microscopic analysis.

ELISA assay: Supernatants from cell cultures were collected and measuredfor the level of mouse cytokines CXCL1 and TNFα using Duoset ELISA kits(R&D system) according to manufacturer's instructions.

RNA-binding assays RIP: The ability of Act1 to bind to RNA in vivo wasassessed as described previously (Datta et al., 2008). Briefly, 10×106Act1−/−MEFs reconstituted by retroviral infection with M2-tagged mousewild-type Act1 (WT) were left untreated or treated with IL-17 (50 ng/ml)for 1 hour. Cells were trypsinized, washed twice, and resuspended in 10ml ice-cold PBS. Cells were fixed in 0.1% formaldehyde for 15 min atroom temperature, whereupon the cross-linking reaction was stopped withglycine (pH 7; 0.25 M). The cells were then washed twice with ice-coldPBS, resuspended in 2 ml RIPA buffer (50 mM Tris-HCl [pH 7.5], 1%Nonidet P-40, 0.5% sodium deoxycholate, 0.05% SDS, 1 mM EDTA, 150 mMNaCl, and pro-teinase inhibitors), and sonicated. The lysate wascentrifuged (15 min, 4° C., 16,000×g), and 1 ml each supernatant wasimmunoprecipitated overnight at 4° C., using Dynabeads (invitrogen)preincubated with 20 ug anti-M2 or anti-IgG Ab. The beads were washedfive times with 1 ml RIPA buffer and resuspended in 150 ul elutionbuffer (50 mM Tris-Cl [pH 7], 5 mM EDTA, 10 mM DTT, 1% SDS).Cross-linking was reversed by incubation at 70° C. for 45 min, RNA waspurified from immunoprecipitates with Trizol (Invitrogen) according tothe manufacturer's instructions and treated with RNase-free DNase, thecDNAs were synthesized and 10% (two microliters) of the reversetranscriptase product was subjected to quantitative real-time PCR.Primers used for quantitative real-time PCR are listed in Table 3.

TABLE 3 Primers for real time quantitative PCR. SEQ ID NO: mCXCL1F: CTGGCCACAGGGGCGCCTATC 62 R: GGACACCTTTTAGCATCTTT 63 mCSF2F: GGCCTTGGAAGCATGTAGAGG 64 R: GGAGAACTCGTTAGAGACGACTT 65 mTNFαF: CATCTTCTCAAAATTCGAGTGACAA 66 R: TGGGAGTAGACAAGGTACAACCC 67 mGAPDHF: GCCTTCCGTGTTCCTACCC 68 R: TGCCTGCTTCACCACCTTC 69 mβ-actinF: GGTCATCACTATTGGCAACG 70 R: ACGGATGTCAACGTCACACT 71 hCXCL1F: AACCGAAGTCATAGCCACAC 72 R: GTTGGATTTGTCACTGTTCAGC 73 hCSF2F: CACTGCTGCTGAGATGAATGAAA 74 R: GTCTGTAGGCAGGTCGGCTC 75 hTNFαF: TCAGCAAGGACAGCAGAG 76 R: GTATGTGAGAGGAAGAGAACC 77 hβ-actinF: GTCGGTATGGGTCAGAAAG 78 R: CTCGTTGTAGAAGGTGTGG 79RIP data analysis: Ct value of each RIP RNA fractions was normalized tothe Input RNA fraction Ct value for the same qPCR Assay (ΔCt) to accountfor RNA sample preparation differences. Then the normalized RIP fractionCt value (ΔCt) was adjusted for the normalized background (anti-IgG)[non-specific (NS) Ab] fraction Ct value (ΔΔCt). The fold enrichment[RIP/non-specific (NS)] was calculated by linear conversion of the ΔΔCt.Below are the formulas used for the calculation: ΔCt [normalized RIP]=Ct[RIP]-(Ct [Input]-Log 2 (fraction of the input RNA saved))); ΔΔCt[RIP/NS]=ΔCt [normalized RIP]-ΔCt [normalized NS]; Fold Enrichment=2(−ΔΔCt [RIP/NS]).

Immunoblot, immunoprecipitation and nuclear fractionation: Cell wereharvested and lysed on ice in a lysis buffer containing 0.5% TritonX-100, 20 mM Hepes pH 7.4, 150 mM NaCl, 12.5 mM-glycerophosphate, 1.5 mMMgCl2, 10 mM NaF, 2 mM dithiothreitol, 1 mM sodium orthovanadate, 2 mMEGTA, 20 mM aprotinin, and 1 mM phenylmethylsulfonyl fluoride for 20minutes, followed by centrifuging at 12,000 rpm for 15 minutes toextract clear lysates. For immunoprecipitation, cell lysates wereincubated with 1 μg of antibody and A-sepharose beads at 4 degreeovernight. After incubation, the beads were washed four times with lysisbuffer and the precipitates were eluted with 2× sample buffer. Elutesand whole cell extracts were resolved on SDS-PAGE followed byimmunobloting with antibodies. Nuclear fractionation was performed usingNUCLEI EZ PREP kit purchased from Sigma in accordance with themanufacturer's instruction. Nuclear pellets were suspended in 30p1 ofnuclear extraction buffer (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mMEGTA in water, pH 7.9) containing freshly prepared 1 mM DTT and proteaseinhibitor cocktail. After 1.5 h incubation on ice bath with intermittentvortexing, extracts were centrifuged and supernatant was collected forimmunoprecipitation.

Expression and purification of His-IL-17RA SEFIR, His-MBP-mAct1-SEFIRand His-MBP proteins: The cDNA encoding a SEFIR domain-containingfragment of human IL17RA (His-IL-17RA SEFIR, aa residues 351 to 616) wassubcloned into a modified pET-28 vector, with a N-terminal 6×His tag anda tobacco etch virus protease (TEV) recognition site (ENLYFQG). TheIL17RA SEFIR domain was expressed and purified bydouble-Nickel-Nitrilotriacetic Acid (Ni-NTA) affinity methods aspreviously described (Deng et al., 2004). Size exclusion chromatographyon a superdex s200 high resolution column was used as a final step forpurification. The cDNA encoding a SEFIR domain containing a fragment ofmouse Act1 (aa residues 391 to 537) and its deletion mutants of theSEFIR domain, designated SEFIR1 to SEFIR5 (based on the five exons thatencode regions of the SEFIR domain: 410 to 439, 440 to 462, 463 to 501,502 to 526, and 527 to 552) was cloned into a modified pET28b vectorthat expresses maltose-binding protein (MBP) with an N-terminal 6×Histag and a C-terminal TEV recognition site. Recombinant mAct1-SEFIR orMBP protein was expressed and purified as previously described (Deng etal., 2008). Size exclusion chromatography on a superdex s200 highresolution column was used as a final step for purification.

In vitro kinase assay: Recombinant IKKi and TBK1 (100 nM) was incubatedrespectively with purified SF2 and Dcp1(10 nM) in the kinase assaybuffer containing 25 mM Tris (pH 7.5), 5 mM β-glycerophosphate, 2 mMDTT, 0.1 mM Na3VO4, 10 mM MgCl2 supplemented with 100 nM ATP and 1 ul[γ-32P]-ATP (PerkinElmer) (10 μCi) at 37° C. for 30 min. The sampleswere subjected to SDS-PAGE followed by authoradiograph.

mRNA decay assay: Endogenous mRNA half-lives were determined with theuse of actinomycin D (5 mg/mL) to inhibit transcription. Pulse-ChasemRNA decay assay in HeLa Tet-off cells were performed as describedpreviously (Datta et al., 2010). Reporter RNA construct for this assaywas obtained by cloning CXCL1 220 (720-940) into pTRE2 (Clontech)containing 5′UTR and coding region of CXCL1 as described previously(KCΔ4; Datta et al., 2010). Total RNA was isolated by TRIzol reagent(Invitrogen) following the manufacturer's instruction, followed byRT-PCR. The values were normalized to the stable (3-actin mRNA.

Statistical analyses: Statistical analyses were performed byMann-Whitney test or by the Student's t test, where appropriate.

Results IL-17 Induces Distinct Act1-RNPs in the Nucleus and CytoplasmicGranules

Messenger RNAs that are not engaged in translation can aggregate intocytoplasmic mRNP granules referred to as P-bodies and stress granules(Anderson et al., 2015; Erickson and Lykke-Andersen, 2011). IL-17stimulation induced the assembly of Act1, the key adaptor of IL-17R,into microscopically visible cytoplasmic granules and nuclearlocalization [FIG. 1A and (Velichko et al., 2016)]. Act1 wasco-localized with Dcp1 decapping enzyme, a component of P-bodies (FIG.1B); but not with TIA1, a marker for stress granules (FIG. 1B). As anegative control, an unrelated signaling molecule IRAK1,interleukin-1-associated kinase 1, was also co-expressed with Dcp1,which failed to co-localize with Dcp1 decapping enzyme. A strong signalwas detected for the interaction between Act1-Dcp1 by in situ ProximityLigation Assay (PLA, which detects direct protein-protein interaction)in response to IL-17 stimulation, indicating the close proximity of Act1with Dcp1 (FIG. 1C). We further validated the Act1-Dcp1 interaction byco-immunoprecipitation in response to IL-17 stimulation (FIG. 1D). Thefact that RNase pretreatment of the lysates abolished the detection ofAct1-Dcp1 interaction suggests that the Act1-Dcp1 interaction isRNA-dependent (FIG. 1D). These results implicate the possible role ofIL-17-Act1 axis in mRNA metabolism in the P-bodies.

We examined whether SF2 and HuR are in the Act1-Dcp1 cytoplasmicgranules. Interestingly, we found that SF2 and HuR were neitherco-localized, nor co-immunoprecipitated with Dcp1 (FIG. 1E). On theother hand, Act1-SF2 and Act1-HuR interaction were indeed detected byPLA in the nucleus and cytoplasmic granules, respectively (FIG. 1F-G).These results indicate that Act1's interaction Dcp1 in the P-bodies islikely independent from the Act1-SF2 and Act1-HuR complexes.Importantly, similar to the Act1-Dcp1 interaction, RNase pretreatment ofthe lysates abolished the detection of Act1-SF2 and Act1-HuRinteraction, suggesting that the Act1's interaction with SF2 and HuRwere also RNA-dependent (FIG. 1D). Taken together, these resultsimplicate the IL-17-Act1 axis in mRNA metabolism via formation ofdistinct Act1-containing RNPs, including Act1-SF2 (RNP1), Act1-Dcp1(RNP2) and Act1-HuR (RNP3).

SEFIR Domain of Act1 is Required for the Formation of Act1-RNPs

Deletion analysis showed the SEIFR domain of Act1 was necessary andsufficient for Act1 to assemble into granules and co-localize with Dcp1(FIG. 1H). We then analyzed the internal deletion mutants of the SEFIRdomain, designated SEFIR1 to SEFIR5 (based on the five exons that encoderegions of the SEFIR domain: 410 to 439, 440 to 462, 463 to 501, 502 to526, and 527 to 552). Deletion of SEFIR2, 3 or 4 had no impact on Act1'sco-localization with Dcp1. Although SEFIR3 (463-501 aa) of Act1 isrequired for its interaction with IL-17R (Liu et al., 2011), ΔSEFIR3 wasstill localized to the P-bodies (FIG. 1H). While ΔSEFIR5 showed reducedco-localization with P-bodies, ΔSEFIR1 completely failed to reside inthe P-bodies (FIG. 1H and Supple. FIG. 2 ). Consistently, ΔSEFIR1 alsofailed to interact with Dcp1 in the co-immunoprecipitation experiment(FIG. 10 . However, both ΔSEFIR1 and ΔSEFIR5 retained their ability tointeract with IL-17R (FIG. 2E). We then examined the Act1's SEFIRmutants for their ability to form Act1-SF2 and Act1-HuR RNPs. Whiledeletion of SEFIR1 impaired the interaction with SF2, ΔSEFIR1 also hadmuch reduced binding to HuR (FIG. 1I). These results indicate that theSEFIR domain of Act1 is required for the formation of Act1-RNPs.

Act1 Directly Binds to the CXCL1 3′UTR Through the SEFIR Domain

Since the SEFIR1, and SEFIR5 to a lesser extent, are required for theformation of Act1-RNPs, one question is how these SEFIR subregions areinvolved in the formation of Act1-RNPs. We modeled the SEFIR domain ofAct1 by SWISS-Model, using the crystal structure of IL-17RA-SEFIR as atemplate. Interestingly, helix αA from SEFIR1 and helix αE from SEFIR5are located in close proximity in Act1 SEFIR domain forming a positivelycharged surface (FIG. 2Aa-b). On the other hand, the corresponding helixαA and helix αE from IL-17RA are not located on the same surface and thehelix αE is negatively charged (FIG. 2Ac-d). Protein surfaces mediatingprotein-RNA interactions are often characterized by positiveelectrostatic potential to complement the negatively charged phosphategroups in the RNA. Thus, it is possible that the SEFIR1+ SEFIR5positively charged surface in Act1 might provide an interactioninterface with negatively charged mRNAs. We speculate that Act1 SEFIRmay directly bind to IL-17-induced mRNAs, which may then permitAct1-targeted stabilization and translation of the bound mRNAs.

To test such a possibility, we subjected Act1 SEFIR and IL-17RA SEFIR toRNA electrophoretic mobility shift assay (REMSA) on CXCL1 3′UTR, whichwas shown to be regulated by the IL-17-Act1-axis for stabilization ofCXCL1 mRNA (Hartupee et al., 2007). Purified recombinant Act1 SEFIR(rkvfitysmdtamevvkfvnfllvngfqtaidifedrirgidiikwmerylrdktymiivaispkykqdvegaesqldedehglhtkyihrmmqiefisqgsmnfrfipvlfpnakkehvptwlqnthvyswpknkknillrllree; SEQ ID NO:95) or IL-17RA SEFIR wasincubated with a radiolabeled RNA probe corresponding to nt 720-940 ofthe CXCL1 3′ UTR (CXCL1 220), which contains IL-17-sensitive motifs,(Datta et al., 2010). The protein-RNA complexes were then separated on anon-denaturing polyacrylamide gel. We found that Act1 SEFIR, but notIL-17RA SEFIR, bound to the CXCL1 probe with Kd=50.2±7.8 nM (FIG. 2B).The binding of Act1 SEFIR to CXCL1 mRNA was specific since the RNA probecorresponding to the GC-rich 3′ UTR of Glutathione Peroxidase 4 (GPx4)remained unbound even at high Act1 SEFIR concentrations. While ΔSEFIR2,ΔSEFIR3 and ΔSEFIR4 retained the ability to bind RNA, ΔSEFIR1 andΔSEFIR5 had much reduced binding to RNA (FIG. 2C-D), suggesting thatSEFIR1 and SEFIR5 are indeed involved in direct binding of Act1 to theCXCL1 mRNA. Mutation of the positively charged amino acid residues inSEFIR1 (K407A) and SEFIR5 (K524A, K526A, and K527A) substantiallyreduced the binding of Act1 SEFIR to RNA, which supports the proposedRNA interaction interface of Act1 SEFIR in FIG. 2Ab. The impaired RNAbinding of ΔSEFIR1 and ΔSEFIR5 correlated with their inability to enterthe P-bodies and form the RNPs (FIG. 1H), which is consistent with theRNA-dependent Act1's interaction with Dcp1, SF2 and HuR shown in FIG.1D.

Act1 Mediates mRNA Stabilization of CXCL1, GM-CSF and TNF Via DirectBinding to their 3′UTRs

Consistent with the fact that ΔSEFIR1 still retained its interactionwith the IL-17R (FIG. 2E), ΔSEFIR1 was able to mediate IL-17-inducedNFkB and JNK activation as wild-type Act1 (FIG. 2F). However,IL-17-induced gene expression, including CXCL1, GM-CSF and TNF, wassubstantially reduced in MEFs transduced with ΔSEFIR1 compared towild-type Act1, at both the mRNA and protein levels (FIG. 2G). SinceAct1 is necessary for IL-17-mediated stabilization of these otherwiseunstable mRNAs of pro-inflammatory genes, we examined whether ΔSEFIR1might be defective in mediating mRNA stabilization. We transfected HeLacells stably expressing tetracycline trans-activator protein (HeLaTet-Off) with a reporter construct pTRE2 CXCL1 containing the regionencoding CXCL1 and a truncated 3′ UTR (nt 720-940, CXCL1 220) thatconfers instability and IL-17-Act1-induced stabilization (Datta et al.,2010). CXCL1 mRNA was readily detected and decayed rapidly with ahalf-life of 30-40 min after transcriptional blockade by the addition ofdoxycycline (Datta et al., 2010). While transfection of wild-type Act1extended the CXCL1 mRNA half-life to 2-3 h, ΔSEFIR1 and ΔSEFIR1 failedto stabilize CXCL1 mRNA as did the vector control (FIG. 2H). Theseresults indicate that the impaired RNA binding of ΔSEFIR1 to CXCL1 3′UTR correlated with its inability to stabilize CXCL1 mRNA.

Since ΔSEFIR1 also lost the ability to mediate IL-17-induced expressionof GM-CSF and TNF, we examined the possible binding of Act1 SEFIR totheir 3′ UTRs. Act1 SEFIR but not ΔSEFIR1 bound the GM-CSF 3′UTR (nt716-1010) and TNF 3′UTR (nt 1362-1507) with similar affinity as theCXCL1 3′ UTR (FIG. 2I). To assess RNA binding of Act1 in the cells, weimmunoprecipitated Act1 from MEFs untreated or treated with IL-17,followed by RT-PCR analysis. Enriched CXCL1, GM-CSF and TNF mRNAs weredetected in Act1 immunoprecipitates compared to that from IgGimmunoprecipitation control (FIG. 2J), suggesting IL-17-induced Act1-RNAbinding in the cells. To examine the functional impact of Act1-RNAbinding, we treated Act1-deficient MEFs reconstituted with wild-typeAct1 or ΔSEFIR1 with TNF (for 0.5 h) to promote transcription ofinflammatory genes, followed by treatment with actinomycin D (to blocktranscription) along with IL-17 (to induce mRNA stabilization). TheCXCL1, GM-CSF and TNF mRNAs were induced to similar levels inAct1-deficient MEFs reconstituted with wild-type Act1 or ΔSEFIR1 afterthe initial treatment with TNF. However, the CXCL1, GM-CSF and TNF mRNAsdecayed more rapidly in Act1-deficient MEFs reconstituted with ΔSEFIR1than that with wild-type Act1 (FIG. 2K). The fact that ΔSEFIR1 failed tobind the 3′ UTRs of CXCL1, GM-CSF and TNF (FIG. 2I) helps to explain theloss of IL-17-mediated stabilization of those mRNAs (FIG. 2K),implicating the critical role of Act1-RNA binding in mRNA stabilization.

Act1 SEFIR Binds to a Stem-Loop Structure in CXCL1 3′UTR

We then aimed to further define the sequences on CXCL1 3′ UTR that arerecognized and bound by Act1 SEFIR. Act1 SEFIR bound nt 720-940 (CXCL1220, FIG. 2A) and nt 780-900 (CXCL1 120) of CXCL1 3′ UTR with similaraffinity (3A-B). Secondary structure prediction (RNAfold web server)indicated that nt 780-900 might form a secondary structure with fourstem-loops (named as A-D, FIG. 3B). By generating sequential deletionsfrom both ends, we narrowed down the Act1-binding region to nt 800-870(CXCL1 70, FIG. 3A-B), which contains an evolutionally conserved region(nt 810-857, CXCL1 47) retaining stem-loop B and C (FIG. 3A).Interestingly, Act1 SEFIR was indeed able to bind to this conserved 47nt sequence (nt 810-857, CXCL1 47), which was then designated as SEFIRbinding element (SBE) (FIGS. 3A and 3C). The binding of Act1 with SBEwas confirmed by Surface Plasmon Resonance which revealed a Kd of 59.5nM.

We then examined the relative importance of stem-loop B and stem-loop Cin SBE's binding to Act1 SEFIR. While the disruption of stem-loop B hadno impact on Act1 SEFIR's binding, impairment of stem-loop C completelyprevented the binding of Act1 SEFIR (FIG. 3C). To test the functionalimportance of stem-loop C, we disrupted stem-loop B and C respectivelyin the 3′UTR of CXCL1 of the reporter construct pTRE2 CXCL1 containingthe region encoding CXCL1 and a truncated 3′ UTR (nt 720-940, CXCL1 220)that confers instability and IL-17-Act1-induced stabilization (Datta etal., 2010). In HeLa Tet-Off cells transfected with pTRE2 CXCL1 orstem-loop B mutant, IL-17 stimulation was able to attenuate CXCL1 mRNAdecay after transcriptional blockade by the addition of doxycycline(FIG. 3D). However, IL-17 stimulation failed to attenuate CXCL1 mRNAdecay in HeLa Tet-Off cells transfected with stem-loop C mutant (FIG.3D). Taken together, our structure-function data implicate that Act1SEFIR's binding to stem-loop C in the 3′ UTR of CXCL1 is required forIL-17-induced CXCL1 mRNA stabilization.

In order to directly map the Act1-SEFIR binding site, we performedenzymatic RNA footprinting on the Act1 SEFIR-SBE complex. Nucleotidesinvolved in the Act1-SBE interaction were identified through partialdigestion of the SBE RNA, which was performed in the absence or presenceof Act1-SEFIR. The native RNA and RNA:protein complexes were thenpartially digested with different ribonucleases and analyzed byelectrophoresis. The cleavage results with the native RNA (Figure. 3E,left panel) are consistent with the predicted structure shown in FIG.3C. RNase A, which cleaves at single-stranded C and U bases, cleaved atU13, U22, U23, U26, U33 and U41-44 (labeled as yellow in Figure. 3F).RNase T1, which cleaves after single-stranded G bases, cleaved G14, G21,G24 and G25 (labeled as purple in Figure. 3F). RNase V1, which cleavesin double-stranded regions, cleaved at multiple positions in stem B andstem C (labeled as blue in FIG. 3F). Several nucleotides (U19, C31, andU33) were cleaved with both single-stranded and double-strandednucleases, suggesting that these regions may breathe. When the SBE wasincubated with Act1-SEFIR, we observed partial protection of specificnucleotides from cleavage by RNase A (U36, U41-44); and RNase V1 (27-29,C31, 36-40) (boxed in FIG. 3E, right panel). Minor protection betweennucleotides 19-23 was occasionally observed but this was not consistentbetween experiments. Interestingly, bases U10-12 became more accessibleto RNase A cleavage which suggests that Act1 binding to stem loopC maycause conformational changes in stem loop B (bracket in FIG. 3E).

Taken together, the footprinting results validated that stem-loop C isthe contact site for Act1 SEFIR. Disruption of the stem in stem-loop C(replacing CCC to GGG) abolished the binding of Act1 SEFIR to RNA,whereas replacement of the sequence in the stem of stem-loop C did notalter the binding of Act1 SEFIR, indicating that it is the secondarystructure rather than the primary sequence that plays a critical rolefor Act1 SEFIR's recognition (FIG. 3G). However, the replacement of theentire loop TAA of the stem-loop C with AGU (without affecting theformation of the stem-loop) also abolished the SEFIR binding,demonstrating the importance of loop sequence for Act1 SEFIR-RNA binding(FIG. 3G).

Act1-RNA Binding to 3′UTR Inhibits Decapping

The next question is how Act1-RNA binding in Dcp1-containing P-bodiesmediates IL-17-induced stabilization of otherwise unstable mRNAs ofpro-inflammatory genes. Notably, the processes of translation and mRNAdegradation are actually coupled (Hu et al., 2009; Mukherjee et al.,2012). Initiation of translation usually involves the interaction oftranslation initiation factors with the 5′ terminal m7G cap that ispresent on most mammalian mRNAs. mRNAs are decapped by the Dcp1/Dcp2decapping enzymes and then degraded 5′ to 3′ by the exonuclease Xrn1(She et al., 2008; Wang et al., 2002). Since both co-immunoprecipitationand in situ PLA showed that Act1 binds to Dcp1, we hypothesized Act1 isspecifically bound to its mRNA targets residing in the P-bodies whereAct1 attenuates decapping via interaction with Dcp1. To test thishypothesis, we examined whether Act1 binding to Dcp1 will affectdecapping activity of Dcp1/Dcp2 complex. Purified Dcp1/Dcp2 and Act1from transfected HeLa cells were incubated with the capped CXCL1 3′UTR(nt 720-940). There was indeed dose-dependent inhibitory effect of Act1on decapping efficiency of Dcp1/Dcp2 complex (FIG. 4Aa, 4B). ΔSEFIR1failed to attenuate decapping, which might be due to its inability tobind CXCL1 3′ UTR (FIGS. 4Ab and 4B). To confirm the importance ofAct1-RNA binding for the Act1's inhibition of decapping, we disruptedthe stem-loop C in cap-labeled fragment of CXCL1 3′UTR (nt 720-940,CXCL1 220) and found that Act1 can no longer block decapping of thismutant reporter transcript (FIGS. 4Ac and 4B).

Act1 Brings TBK1 to Phosphorylate Dcp1 that Dissociates from Dcp2

It was shown that Dcp1 phosphorylation at S315 is required for theinactivation of the decapping activity (Aizer et al., 2013; Rzeczkowskiet al., 2011). Using anti-p-5315 antibody, we found that IL-17 treatmentindeed induced Dcp1 phosphorylation at S315. IL-17-induced Dcp1phosphorylation was impaired in Act1-deficient cells (FIG. 4C). Recentstudies reported two Act1-interacting kinases: IKKi and TBK1(Bulek etal., 2011; Qu et al., 2012). Thus, we tested whether these kinases areinvolved in Act1-mediated regulation of Dcp1. Throughimmunoprecipitation we confirmed that both IKKi and TBK1 interacted withAct1 upon IL-17 stimulation (FIG. 4D). Interestingly while both IKKi andTBK1 colocalized with Act1, only TBK1 showed colocalization with Dcp1.Consistently, IL-17 stimulation induced the interaction of TBK1 withDcp1, which was abolished in Act1-deficient cells (FIG. 4C), indicatingthat IL-17-induced TBK1-Dcp1 interaction is Act1-dependent. WhileIL-17-induced Dcp1 phosphorylation (but not p-JNK or p-p65) wasdiminished in TBK1-deficient cells, IKKi deficiency had little effect(FIG. 4E). Recombinant TBK1 was able to phosphorylate Dcp1 in an invitro kinase assay, implicating that TBK1 might be a direct kinase forDcp1 (FIG. 4F). Consistently, TBK1 overexpression diminished theinteraction between Dcp1 and Dcp2, while TBK1 failed to remove Dcp1S315A mutant from Dcp2 (FIG. 4G). Interestingly, we observed thatIL-17-induced Act1-Dcp1 interaction was enhanced and more sustained inthe presence of TBK1 inhibitor, confirming the impact of TBK1 kinaseactivity on the dissociation of Dcp1 from the Act1-RNP complex (FIG.4H). Based on these findings, we propose that Act1 brings TBK1 to themRNA targets in the P-bodies where TBK1 phosphorylates Dcp1, resultingin Dcp1 dissociation from Act1-RNP, inhibition of decapping andstabilization of mRNAs. In support of this, TBK1 inhibitor effectivelyabolished Act1-mediated inhibition of decapping on cap-labeled fragmentof CXCL1 3′UTR (nt 720-940, CXCL1 220) (FIGS. 4Ad and 4B). Westernanalysis confirmed that TBK1 was indeed co-purified with Act1 used forthe decapping assay. Consistently, we found TBK1 deficiency indeedsubstantially reduced IL-17-induced expression of CXCL1, GM-CSF and TNFat both mRNA and protein levels (FIG. 4I).

Act1 Forms Distinct RNPs with Dcp1/Dcp2, SF2 and HuR

Our results described here have defined an Act1-RNP consisting ofAct1-TBK1-Dcp1/2, which was designated as RNP2. While imaging studiesindicated that SF2 and HuR were not co-localized with Dcp1 (FIG. 1F), weperformed immunoprecipitation experiments to carefully examine thedifferent Act1-RNPs. By co-immunoprecipitation with anti-Dcp1 usinglysates from untreated and IL-17-treated Act1-expressing MEFs, we showedthat while IL-17 induced Dcp1-Act1-TBK1 interaction in an RNA-dependentmanner (sensitive to RNase treatment), SF2 and HuR were not detected inthis RNP (RNP2). IL-17-induced Act1-SF2 complex (RNP1) was alsosensitive to RNase pretreatment, but Dcp1-TBK1 and HuR were not found inthis Act1-SF2-RNP (Supple FIG. 1B). Likewise, we failed to detectDcp1-TBK1 and SF2 in IL-17-induced Act1-HuR RNP (RNP3). Taken together,these results suggest that the Act1-SF2, Act1-Dcp1/2 and Act1-HuRrepresent three independent RNPs. Importantly, deletion of SEFIR1(ΔSEFIR1) abolished the formation of all three RNPs, implicating theimportance of Act1's binding to mRNA for the formation of theseAct1-RNPs (Supple FIG. 1A-C). In support of this, PLA imaging showedthat deletion of SEFIR1 (ΔSEFIR1) also impaired the Act1-SF2 interaction(in the nucleus) and Act1-HuR interaction (in the cytosol) as well (FIG.5A-B).

SF2 has been shown to mediate decay of cytokine and chemokine mRNA. Itwas reported that SF2 bound chemokine mRNA (induced by TNF) in theabsence of IL-17 stimulation (Sun et al., 2011), whereas the SF2-mRNAinteraction was much reduced after stimulation with IL-17 in anAct1-dependent manner (FIG. 5C). Interestingly, we now found that IL-17failed to reduce SF2's binding to CXCL1 in Act1-deficient cells restoredwith ΔSEFIR1, suggesting that Act1's RNA binding might be required forthe dissociation of SF2 from the mRNAs (FIG. 5C). One important questionis how Act1's binding to mRNAs promotes the dissociation of SF2 from themRNAs. We examined the sequences of CXCL1 required for SF2 binding.Surprisingly, we found that SF2 was able to bind the same region ofCXCL1 as Act1: the SEFIR binding element (SBE); and the impairment ofstem-loop C completely prevented the binding of SF2 (FIG. 5D-E).Addition of increasing amounts of Act1 to the RNA binding reactionattenuated SF2's binding to SBE (FIG. 5F). Notably, SF2 phosphorylationhas been implicated as a mechanism for its dissociation from mRNAtargets (Huynh et al., 2009). While both imaging and fractionationexperiments indicated that Act1-SF2 RNP1 resides in the nucleus (FIGS.5A-B and 5G), IKKi (a kinase required for IL-17-mediated mRNAstabilization (Bulek et al., 2011) was translocated into the nucleus anddetected in the Act1-SF2 RNP1 in response to IL-17 stimulation (FIG.5G). Importantly, IKKi indeed was able to phosphorylate SF2 in vitro(FIG. 5H) and incubation of recombinant IKKi with SF2 reduced theability of SF2 to bind CXCL1 mRNA (FIG. 5I), implicating IKKi inpreventing SF2's binding to mRNAs. Whereas IL-17-induced IKKi-SF2 andAct1-SF2 interaction were abolished in Act1-deficient MEFs restored withΔSEFIR1 (FIG. 5C and FIG. 5G), Act1-IKKi interaction in both cytosol andnucleus was retained in these cells (FIG. 5G). These results suggestthat IL-17 induces Act1-IKKi interaction prior to their translocation tothe nucleus and Act1's binding to SF2-bound mRNAs in the nucleus allowsIKKi to phosphorylate SF2, attenuating SF2's binding to the mRNAtargets.

On the other hand, HuR was implicated in shifting target mRNAs topolysomes for protein translation and IL-17 stimulation induced theco-shift of Act1-HuR to the polysomes (Herjan et al., 2013; Tiedje etal., 2012). Notably, IL-17 induced the binding of HuR to CXCL1 inAct1-expressing MEFs, which was abolished in Act1-deficient MEFsrestored with ΔSEFIR1, suggesting that Act1's RNA binding might also berequired for HuR's recruitment to the target mRNAs (FIG. 5C). Weexamined the sequences of CXCL1 required for HuR binding and found thatHuR bound to CXCL1 220-I (nt 720-830), but not CXCL1 220-II (nt 830-940)(FIGS. 5D and 5J). Interestingly, we found HuR and Act1 cansimultaneously bind to CXCL1 220 (nt 720-940) (FIGS. 5D and 5K). Removalof nt 720-829 from CXCL1 220 abolished HuR binding, whereas Act1 bindingwas retained [CXCL1 220-II (nt 830-940)], indicating that HuR and Act1have their independent binding sequences on CXCL1 (FIGS. 5D and 5K).These results indicate that besides blocking decapping in RNP2(Act1-TBK1-Dcp1/2), Act1-RNA binding has additional functions by formingRNP1 (Act1-SF2) and RNP3 (Act1-HuR). Since HuR is not part of RNP1(Act1-SF2) (FIG. 5A-C), Act1 may facilitate HuR's binding to CXCL1 invivo after competing off SF2 from target mRNAs. In support of this,IL-17-induced the co-shift of Act1-HuR to the polysomes (Herjan et al.,2013) was abolished in Act1-deficient MEFs restored with ΔSEFIR1 (FIG.5L-M). Consistently, the ratio of CXCL1 mRNA in the translation activepolysomes over the inactive fractions was substantially reduced inAct1-deficient MEFs restored with ΔSEFIR1 (FIG. 5M-N). Based on theseresults, we propose the following model for the actions of Act1-RNAbinding (FIG. 5O): First, IL-17 induces Act1's binding to the mRNAs(such as CXCL1) in the nucleus (RNP1) preventing SF2-mdiated mRNA decayby competing off SF2's binding to the mRNAs which is further promoted byIKKi-mediated SF2 phosphorylation. Second, Act1 follows the mRNAs to theP-bodies (RNP2) inhibiting Dcp1/2-mediated mRNA decapping by employingTBK1 to phosphorylate Dcp1. Lastly, Act1-mRNAs are shifted to thepolysomes by facilitating HuR's binding to mRNAs (RNP3) for proteintranslation.

SBE RNA Aptamers Abolished IL-17-Induced mRNA Stabilization of CXCL1,GM-CSF and TNF

Our results suggest that Act1 directly binds to the 3′UTRs ofinflammatory genes, forming Act1-RNPs to inhibit mRNA decay and promoteprotein translation. Based on these findings, we hypothesized that RNAoligonucleotides corresponding to the SBE (SEFIR Binding Element) mightinhibit the effect of Act1 in the defined RNPs and inflammatory geneexpression. Interestingly, SBE RNA aptamers with or without mutation instem-loop B was indeed able to compete off Act1 SEFIR's binding to theCXCL1 3′UTR (FIG. 6A). On the other hand, SBE RNA aptamer with mutatedstem-loop C failed to compete off Act1 SEFIR's binding to CXCL1 3′UTR(FIG. 6A). To test the inhibitory effect of SBE RNA aptamers in cellculture and in vivo models, we generated fluorescently-labeled andpartially modified SBE RNA aptamers where first three 5′ and three 3′nucleotides were methylated (2′-OH). We co-transfected the fluorescentSBE RNA aptamers with SF2-HA or Act1-RFP into HeLa cells. Thetransfected fluorescent SBE RNA aptamers mainly resided in the cytoplasmand was detected in Act1 granules (FIG. 6B). Interestingly, we observedthat the Act1 granules were substantially reduced over time in cellstransfected with SBE RNA aptamers, implicating that this SBE RNAaptamers might be disrupting the Act1's co-localization with theP-bodies (FIG. 6B). We indeed found that SBE RNA aptamers, but not SBEaptamers with mutated stem-loop C, abolished the formation of Act1granules and Act1's co-localization with Dcp1 (FIG. 6C). These resultssuggest that SBE RNA aptamers-mediated inhibition of Act1's RNA bindingactivity may affect other transcripts in addition to CXCL1. In supportof this, the SBE RNA aptamers was also able to compete off Act1 SEFIR'sbinding to the 3′ UTRs of GM-CSF and TNF (FIG. 6D and Supple. FIG. 6Bsuggesting that the binding of SBE RNA aptamers to Act1 SEFIR blocksAct1's binding to the mRNA targets. These results suggest that the SBERNA aptamers has the potential to be an inhibitory agent for blockingIL-17-induced inflammatory response. We indeed found that transfectionof SBE RNA aptamers reduced IL-17-mediated mRNA stabilization and theproduction of CXCL1, GM-CSF and TNF (FIG. 6E-F).

SBE RNA Aptamers Inhibited IL-17-Dependent Skin Hyperplasia

Secukinumab (anti-IL-17A) showed great efficacy for psoriasis and hasbeen approved by FDA for treatment of psoriasis. Aberrant keratinocyteproliferation and neutrophilic inflammation are well-known hallmarks ofpathogenesis of psoriasis. To examine the impact of SBE RNA aptamers onIL-17A-induced epidermal proliferation and inflammation, the ears of WTC57BL/6 female mice were injected intradermally with IL-17A with SBE RNAaptamers or SBE mutant aptamers (mutated stem-loop C as a negativecontrol) for 6 consecutive days. We found that SBE RNA aptamer, but notmutant aptamer substantially reduced IL-17A-dependent epidermalhyperplasia and neutrophil infiltration in the ears (FIG. 7A-C).Likewise, SBE RNA aptamer, but not mutant aptamer greatly diminishedIL-17A-induced expression of cxcl1, gm-csf and tnf in the ears (FIG.7D). Taken together, these data indicate that IL-17A induceskeratinocytes proliferation and neutrophil infiltration, resulting inepidermal hyperplasia, which were effectively blocked by SBE RNAaptamers.

The SEFIR domain, a conserved motif present in the cytoplasmic regionsof IL-17 receptor subunits and adaptor Act1, mediates the recruitment ofAct1 to the receptor upon IL-17 stimulation. Here we have unexpectedlyidentified SEFIR of Act1 as a direct RNA binding domain, rendering Act1RNA binding activity to stabilize otherwise unstable mRNAs of thepro-inflammatory genes (CXCL1, TNF and GM-CSF) in response to IL-17stimulation. We found that Act1 directly binds to the 3′-UTRs ofinflammatory mRNAs to form distinct RNPs in several subcellularcompartments including P-bodies controlling mRNA metabolism.Structure-function analysis showed that Act1 SEFIR binds to a stem-loopstructure (named as SBE) in the CXCL1 3′UTR. RNA aptamers containing SBEabolished Act1's binding to the target mRNAs and attenuatedIL-17-mediated mRNA stabilization. The physiologic relevance of theRNA-binding activity of Act1 is illustrated by our discovery that SBERNA aptamers inhibited IL-17-induced skin inflammation. While thepresent invention is not limited to any particular mechanism, and anunderstanding of the mechanism is not necessary to practice theinvention, it is believed that this study demonstrates that thereceptor-proximal adaptor Act1 directly binds to mRNAs to control thesteps of RNA metabolism, which provides a novel molecular mechanism ofinflammatory response via receptor-specific mRNA stabilization of selectinflammatory genes. Moreover, the discovery of a non-canonical functionof Act1 will allow the development of new therapeutic strategies forautoimmune inflammatory diseases.

In addition to mRNAs of inflammatory genes, short half-lives allowdynamic regulation of a wide spectrum of transcripts whose expressionalso needs to be switched on and off rapidly, including transcriptionfactors, signaling components, and cell cycle proteins (Schoenberg andMaquat, 2012). These unstable mRNAs possess destabilizing sequences intheir 3′UTRs (including AU-rich element and Stem-Loops) that arerecognized by RNA binding proteins (ARE and SL binding proteins) tomediate the degradation of the mRNAs via deadenylation, decapping, andexonucleolytic degradation (Mino et al., 2015; Schoenberg and Maquat,2012; Stumpo et al., 2010). While multiple mRNA destabilizing mechanismshave been discovered, it remains unclear how the different classes ofmRNAs with short half-lives are stabilized in a cell context andstimulus specific manner. Although ARE binding protein SF2 and HuR werepreviously implicated in IL-17-induced mRNA stabilization, they cannotexplain the receptor-specific mRNA stabilization of select inflammatorygenes (Brennan and Steitz, 2001; Herjan et al., 2013). Notably, HuR hasa broad range of mRNA targets including transcription factors, signalingcomponents, and cell cycle proteins, whereas IL-17 stimulation does notstabilize all of the HuR mRNA targets (Mitchell and Parker, 2014;Schoenberg and Maquat, 2012; Mukherjee et al., 2011). Likewise, SF2 hasbeen implicated in various aspects RNA metabolism, including RNAsplicing, mRNA export, and nonsense-mediated decay (Cao et al., 1997;Krainer et al., 1990; Lemaire et al., 2002; Reed and Cheng, 2005; Sun etal., 2011; Zhong et al., 2009). Therefore, the discovery of Act1 as adirect RNA binding protein is a conceptual advancement for ourunderstanding how short-lived mRNAs can be stabilized in a stimulusspecific manner via a receptor-mediated direct mechanism.

In eukaryotes, mRNA decay pathways are initiated by deadenylationcarried out by the CCR4-CAF1-NOT deadenylase complex (Chen and Shyu,2011). An exonuclease complex (the exosome) can degrade mRNAs in a 3′-5′direction post deadenylation; and the Dcp1/Dcp2 decapping enzyme exposesthe mRNA to degradation from the 5′ end using the exonuclease Xrn1,simultaneously shutting down translation initiation (Arribas-Layton etal., 2013; Franks and Lykke-Andersen, 2008; Schoenberg and Maquat,2012). Previous studies have shown that the mRNA targeting intodecapping involves the formation of translationally repressed mRNP,which can be targeted to P-bodies and stress granules (Anderson et al.,2015; Arribas-Layton et al., 2013; Franks and Lykke-Andersen, 2008). Wehere found that one of the Act1-containing RNPs is localized in theP-bodies (RNP2, not in the stress granules) and Act1 is able to inhibitthe decapping activity of Dcp1/Dcp2 complex by bringing a kinase, TBK1to phosphorylate Dcp1 and disrupt Dcp1/Dcp2 complex. As a result,Act1-bound mRNAs including CXCL1, TNF and GM-CSF are stabilized andtranslated. Our findings are consistent with the concepts that thetranslation and mRNA decay are in competition with each other and theprocesses of translation and mRNA degradation are coupled. Therefore, wepropose that Act1-RNP-mediated inhibition of decapping releases themRNAs trapped in the P-bodies for return to translation. Supporting thepotential role Act1 in linking mRNA stabilization to proteintranslation, it was previously reported that IL-17 stimulation inducedthe co-shift of Act1-HuR to the polysomes (Herjan et al., 2013).Importantly, we now found that RNase pretreatment of the lysatesabolished the detection of Act1-HuR interaction, suggesting that theAct1's interaction with HuR is RNA-dependent. Consistently, we found HuRand Act1 can simultaneously bind to CXCL1; and IL-17 induced the bindingof HuR to CXCL1 was abolished in Act1-deficient cells restored withΔSEFIR1. These results suggest that Act1's RNA binding is required forHuR's recruitment to the target mRNAs. Based on these findings, weproposed that Act1-bound mRNAs are shifted to the polysomes byfacilitating HuR's binding to mRNAs (RNP3) for protein translation. Insupport of this, we indeed found that IL-17 stimulation induced theco-shift of Act1-HuR to the polysomes was abolished in Act1-deficientcells restored with ΔSEFIR1.

Although cytoplasmic mRNA decay seems to be the dominant pathway formRNA turnover in eukaryotes, recent studies have implicated regulationof mRNA stability in the nucleus. It has been estimated that only aminor proportion, about 30% of transcripts in eukaryotes, is processedto be mRNA and exported to the cytoplasm (Jackson et al., 2000).

While aberrant RNA processing contributes to the trapping the nucleartranscripts in the nucleus, both 3′ to 5′ and 5′ to 3′ exoribonucleaseshave been found in the nucleus (Brannan et al., 2012; Bresson et al.,2017; Gudipati et al., 2012). Therefore, it is conceivable that activeintervention to prevent degradation of nuclear transcripts is requiredfor abundant production and translation of mRNAs. In support of this,while SF2 has been shown to mediate mRNA decay, we found Act1 forms anRNP with SF2 in the nucleus (RNP1). IL-17 stimulation induced thedissociation of SF2 from the mRNA targets, which was abolished inAct1-deficient cells restored with ΔSEFIR1 suggesting that Act1's RNAbinding is required for preventing SF2-dependent mRNA decay.Interestingly, we found that SF2 was able to bind the same region ofCXCL1 as Act1. The addition of increasing amounts of Act1 to the RNAbinding reaction attenuated SF2's binding to the target mRNA, suggestingthat the two proteins directly compete for binding to the same targetmRNA. Additionally, SF2 phosphorylation has been implicated as amechanism for its dissociation from mRNA targets (Cao et al., 1997; Xiaoand Manley, 1997). In support of this, we found that IL-17 inducesAct1-IKKi nuclear translocation and Act1's binding to SF2-bound mRNAs inthe nucleus allows IKKi to phosphorylate SF2, preventing SF2's bindingto the mRNA targets. However, it remains unclear how SF2 mediates mRNAdecay. SF2-mediated mRNA decay might be through active recruitment ofexonucleases. Alternatively, SF2-bound mRNAs may be simply trapped inthe nucleus and are degraded over time. In any case, with the help ofIKKi, Act1 binding to mRNAs drives off SF2, resulting in stabilizationof mRNAs.

Taken together, while the present invention is not limited to anyparticular mechanism, we propose the following model for the actions ofAct1-RNA binding for IL-17-induced inflammatory response. Upon IL-17stimulation, multiple signaling pathways (including NFkB and MAPKs) areactivated to induce the gene transcription of cytokines and chemokines.Act1 then directly binds the mRNAs of cytokines and chemokines tostabilize these otherwise unstable mRNAs for the production of thepro-inflammatory mediators. Act1's binding to mRNAs of inflammatorygenes results in the formation of multiple RNPs controlling differentsteps of mRNA metabolism. First, Act1 binds to the mRNAs in the nucleus(RNP1) inhibiting SF2-mdiated mRNA degradation by competing off SF2'sbinding to mRNAs, which was further facilitated by IKKi-mediated SF2phosphorylation. One of the possible roles of Act1-RNP1 in the nucleusis to protect the degradation and/or trapping of nascent nucleartranscripts. Second, Act1 forms a RNP (RNP2) in the P-bodies blockingDcp1/2-mediated mRNA decapping by recruiting TBK1 to phosphorylate Dcp1.The Act1-RNP2 may represent an action for how to resolve the competitionbetween mRNA translation and degradation. Finally, Act1-mRNAs areco-shifted with HuR to the polysomes (RNP3) for protein translation.Taken together, the study here provides the first example of areceptor-interacting adaptor molecule, Act1, playing a direct role inmRNA metabolism, orchestrating receptor-mediated selectivity of mRNAstabilization and translation. Additionally, it is exciting to find thatSBE RNA aptamers was able to disrupt the co-localization of Act1 withP-bodies.

REFERENCES

-   Aizer, et al., (2013). The P body protein Dcp1a is    hyper-phosphorylated during mitosis. PloS One 8, e49783.-   Anderson, et al., (2015). Stress granules, P-bodies and cancer.    Biochim. Biophys. Acta 1849, 861-870.-   Arribas-Layton, et al., (2013). Structural and functional control of    the eukaryotic mRNA decapping machinery. Biochim. Biophys. Acta    1829, 580-589.-   Brannan, et al. (2012). mRNA decapping factors and the exonuclease    Xrn2 function in widespread premature termination of RNA polymerase    II transcription. Mol. Cell 46, 311-324.-   Brennan, et al., (2001). HuR and mRNA stability. Cell. Mol. Life    Sci. CMLS 58, 266-277.-   Bresson, et al., (2017). Nuclear RNA Decay Pathways Aid Rapid    Remodeling of Gene Expression in Yeast. Mol. Cell 65, 787-800.e5.-   Bulek, et al. (2011). The inducible kinase IKKi is required for    IL-17-dependent signaling associated with neutrophilia and pulmonary    inflammation. Nat. Immunol. 12, 844-852.-   Cao, et al., (1997). Both phosphorylation and dephosphorylation of    ASF/SF2 are required for pre-mRNA splicing in vitro. RNA 3,    1456-1467.-   Chang, et al., (2006). Act1 adaptor protein is an immediate and    essential signaling component of interleukin-17 receptor. J. Biol.    Chem. 281, 35603-35607.-   Chen, et al., (2011). Mechanisms of deadenylation-dependent decay.    Wiley Interdiscip. Rev. RNA 2, 167-183.-   Chesné, et al., (2015). Prime role of IL-17A in neutrophilia and    airway smooth muscle contraction in a house dust mite-induced    allergic asthma model. J. Allergy Clin. Immunol. 135, 1643-1643.e3.-   Cho, et al. (2010). IL-17 is essential for host defense against    cutaneous Staphylococcus aureus infection in mice. J. Clin. Invest.    120, 1762-1773.-   Conti, et al. (2009). Th17 cells and IL-17 receptor signaling are    essential for mucosal host defense against oral candidiasis. J. Exp.    Med. 206, 299-311.-   Cua, et al., (2010). Innate IL-17-producing cells: the sentinels of    the immune system. Nat. Rev. Immunol. 10, 479-489.-   Datta, et al., (2008). Tristetraprolin regulates CXCL1 (KC) mRNA    stability. J. Immunol. Baltim. Md. 1950 180, 2545-2552.-   Datta, et al., (2010). IL-17 regulates CXCL1 mRNA stability via an    AUUUA/tristetraprolin-independent sequence. J. Immunol. Baltim. Md.    1950 184, 1484-1491.-   Deng, et al., (2004). An improved protocol for rapid freezing of    protein samples for long-term storage. Acta Crystallogr. D Biol.    Crystallogr. 60, 203-204.-   Deng, et al., (2008). Structure of the ROC domain from the    Parkinson's disease-associated leucine-rich repeat kinase 2 reveals    a dimeric GTPase. Proc. Natl. Acad. Sci. U.S.A. 105, 1499-1504.-   Erickson, et al., (2011). Cytoplasmic mRNP granules at a glance. J.    Cell Sci. 124, 293-297.-   Franks, et al., (2008). The control of mRNA decapping and P-body    formation. Mol. Cell 32, 605-615.-   Gu, et al., (2013). IL-17 family: cytokines, receptors and    signaling. Cytokine 64, 477-485.-   Gudipati, et al., (2012). Extensive degradation of RNA precursors by    the exosome in wild-type cells. Mol. Cell 48, 409-421.-   Harrington, et al., (2005). Interleukin 17-producing CD4+ effector T    cells develop via a lineage distinct from the T helper type 1 and 2    lineages. Nat. Immunol. 6, 1123-1132.-   Hartupee, et al., (2007). IL-17 enhances chemokine gene expression    through mRNA stabilization. J. Immunol. Baltim. Md. 1950 179,    4135-4141.-   Herjan, et al. (2013). HuR is required for IL-17-induced    Act1-mediated CXCL1 and CXCL5 mRNA stabilization. J. Immunol.    Baltim. Md. 1950 191, 640-649.-   Hu, et al, (2009). Co-translational mRNA decay in Saccharomyces    cerevisiae. Nature 461, 225-229.-   Huynh, et al., (2009). Allosteric Interactions Direct Binding and    Phosphorylation of ASF/SF2 by SRPK1. Biochemistry (Mosc.) 48,    11432-11440.-   Jackson, et al., (2000). The balance sheet for transcription: an    analysis of nuclear RNA metabolism in mammalian cells. FASEB J. 14,    242-254.-   Kang, et al. (2010). Astrocyte-restricted ablation of    interleukin-17-induced Act1-mediated signaling ameliorates    autoimmune encephalomyelitis. Immunity 32, 414-425.-   Kolls, et al. (2010). The role of Th17 cytokines in primary mucosal    immunity. Cytokine Growth Factor Rev. 21, 443-448.-   Krainer, et al., (1990). The essential pre-mRNA splicing factor SF2    influences 5′ splice site selection by activating proximal sites.    Cell 62, 35-42.-   Lemaire, et al., (2002). Stability of a PKCI-1-related mRNA is    controlled by the splicing factor ASF/SF2: a novel function for SR    proteins. Genes Dev. 16, 594-607.-   Leppek, et al., (2013). Roquin promotes constitutive mRNA decay via    a conserved class of stem-loop recognition motifs. Cell 153,    869-881.-   Li, et al., (2000). Act1, an NF-kappa B-activating protein. Proc.    Natl. Acad. Sci. U.S.A. 97, 10489-10493.-   Liu, et al. (2011). A CC′ loop decoy peptide blocks the interaction    between Act1 and IL-17RA to attenuate IL-17- and IL-25-induced    inflammation. Sci. Signal. 4, ra72.-   Liu, et al. (2017). The flavonoid cyanidin blocks binding of the    cytokine interleukin-17A to the IL-17RA subunit to alleviate    inflammation in vivo. Sci Signal 10, eaaf8823.-   Milner, et al., (2013). The cup runneth over: lessons from the    ever-expanding pool of primary immunodeficiency diseases. Nat. Rev.    Immunol. 13, 635-648.-   Mino, et al. (2015). Regnase-1 and Roquin Regulate a Common Element    in Inflammatory mRNAs by Spatiotemporally Distinct Mechanisms. Cell    161, 1058-1073.-   Mitchell, et al., (2014). Principles and properties of eukaryotic    mRNPs. Mol. Cell 54, 547-558.-   Mukherjee, et al., (2012). Identification of cytoplasmic capping    targets reveals a role for cap homeostasis in translation and mRNA    stability. Cell Rep. 2, 674-684.-   Mukherjee, et al., (2011). Integrative regulatory mapping indicates    that the RNA-binding protein HuR couples pre-mRNA processing and    mRNA stability. Mol. Cell 43, 327-339.-   Novatchkova, et al., (2003). The STIR-domain superfamily in signal    transduction, development and immunity. Trends Biochem. Sci. 28,    226-229.-   Park, et al. (2005). A distinct lineage of CD4 T cells regulates    tissue inflammation by producing interleukin 17. Nat. Immunol. 6,    1133-1141.-   Patel, et al., (2013). Effect of IL-17A blockade with secukinumab in    autoimmune diseases. Ann. Rheum. Dis. 72 Suppl 2, ii116-ii123.-   Qian, et al. (2007). The adaptor Act1 is required for interleukin    17-dependent signaling associated with autoimmune and inflammatory    disease. Nat. Immunol. 8, 247-256.-   Qu, et al., (2012). TRAF6-Dependent Act1 Phosphorylation by the IκB    Kinase-Related Kinases Suppresses Interleukin-17-Induced NF-κB    Activation. Mol. Cell. Biol. 32, 3925-3937.-   Reed, et al., (2005). TREX, SR proteins and export of mRNA. Curr.    Opin. Cell Biol. 17, 269-273.-   Ruzankina, et al., (2007). Deletion of the developmentally essential    gene ATR in adult mice leads to age-related phenotypes and stem cell    loss. Cell Stem Cell 1, 113-126.-   Rzeczkowski, et al., (2011). c-Jun N-terminal kinase phosphorylates    DCP1a to control formation of P bodies. J. Cell Biol. 194, 581-596.-   Schoenberg, et al., (2012). Regulation of cytoplasmic mRNA decay.    Nat. Rev. Genet. 13, 246-259.-   She, et al., (2008). Structural basis of dcp2 recognition and    activation by dcp1. Mol. Cell 29, 337-349.-   Shen, et al., (2008). Structure-function relationships in the IL-17    receptor: implications for signal transduction and therapy. Cytokine    41, 92-104.-   Stoecklin, et al., (2006). ARE-mRNA degradation requires the 5′-3′    decay pathway. EMBO Rep. 7, 72-77.-   Stumpo, et al., (2010). Inflammation: cytokines and RNA-based    regulation. Wiley Interdiscip. Rev. RNA 1, 60-80.-   Sun, et al., (2011). Treatment with IL-17 prolongs the half-life of    chemokine CXCL1 mRNA via the adaptor TRAF5 and the    splicing-regulatory factor SF2 (ASF). Nat. Immunol. 12, 853-860.-   Swaidani, et al., (2009). The critical role of epithelial-derived    Act1 in IL-17- and IL-25-mediated pulmonary inflammation. J.    Immunol. Baltim. Md. 1950 182, 1631-1640.-   Tiedje, et al., (2012). The p38/MK2-driven exchange between    tristetraprolin and HuR regulates AU-rich element-dependent    translation. PLoS Genet. 8, e1002977.-   Toy, et al., (2006). Cutting edge: interleukin 17 signals through a    heteromeric receptor complex. J. Immunol. Baltim. Md. 1950 177,    36-39.-   Velichko, et al., (2016). A Novel Nuclear Function for the    Interleukin-17 Signaling Adaptor Protein Act1. PLOS ONE 11,    e0163323.-   Wang, et al., (2002). The hDcp2 protein is a mammalian mRNA    decapping enzyme. Proc. Natl. Acad. Sci. U.S.A. 99, 12663-12668.-   Xiao, et al., (1997). Phosphorylation of the ASF/SF2 RS domain    affects both protein-protein and protein-RNA interactions and is    necessary for splicing. Genes Dev. 11, 334-344.-   Zhang, et al., (2014). Structure of the unique SEFIR domain from    human interleukin 17 receptor A reveals a composite ligand-binding    site containing a conserved α-helix for Act1 binding and IL-17    signaling. Acta Crystallogr. D Biol. Crystallogr. 70, 1476-1483.-   Zhong, et al., (2009). SR proteins in Vertical Integration of Gene    Expression from Transcription to RNA Processing to Translation. Mol.    Cell 35, 1-10.

All publications and patents mentioned in the specification and/orlisted below are herein incorporated by reference. Various modificationsand variations of the described method and system of the invention willbe apparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific embodiments, it should be understood thatthe invention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in therelevant fields are intended to be within the scope described herein.

We claim:
 1. A method of treating an IL-17a related disease or conditioncomprising: treating a subject with an IL-17a related disorder orcondition with a composition, wherein said composition comprises a firstnucleic acid sequence, wherein said first nucleic acid sequencecomprises an SBE nucleic acid sequence that binds a SEFIR domain of anACT1 protein.
 2. The method of claim 1, wherein at least a portion ofsaid SBE nucleic acid sequence is from a gene selected from CXCL1,GM-CSF, and TNF.
 3. The method of claim 1, wherein said SBE nucleic acidsequence comprises a sequence shown in SEQ ID NOs:1-2, 49-61, 88-94, and99-129.
 4. The method of claim 1, wherein said SBE nucleic acid sequencecomprises RNA bases.
 5. The method of claim 1, wherein said SBE nucleicacid sequence comprises DNA bases.
 6. The method of claim 1, whereinsaid treating reduces or eliminates at least one symptom related to saidIL-17a related disease or condition.
 7. The method of claim 1, whereinsaid IL-17a related disease is selected from the group consisting of:psoriasis, chronic plaque, asthma, an autoimmune disease, aninflammatory condition, rheumatoid arthritis, and multiple sclerosis. 8.The method of claim 1, wherein said SBE nucleic acid sequence comprises,consist of, or consists essentially of: nucleotides 810-857 of saidCXCL1 gene, ii) nucleotides 830-856 of said CXCL1 gene, or iii)nucleotides 800-835 of said CXCL1 gene.
 9. The method of claim 1,wherein said subject is human.
 10. The method of claim 1, wherein saidSBE nucleic acid sequence is from a human gene.
 11. The method of claim1, wherein said ACT1 protein is human ACT1 protein.
 12. The method ofclaim 1, wherein said first nucleic acid sequence is between 12 and 70nucleotides in length.
 13. A composition comprising a first nucleic acidsequence, wherein said first nucleic acid sequence comprises an SBEnucleic acid sequence that binds a SEFIR domain of an ACT1 protein, andwherein said first nucleic acid sequence comprises modified bases toimprove stability in vivo.
 14. The composition of claim 13, wherein saidSBE nucleic acid sequence is from a gene selected from CXCL1, GM-CSF,and TNF.
 15. The composition of claim 13, wherein said first nucleicacid sequence is no longer than 70 bases and comprises at least: i)nucleotides 830-856 of said CXCL1 gene, or ii) nucleotides 810-857 ofsaid CXCL1 gene.
 16. The composition of claim 13, wherein said firstnucleic acid sequence is composed of RNA bases.
 17. The composition ofclaim 13, wherein said first nucleic acid is present in said compositionat a level that is therapeutic when administered to a subject with anIL-17a related disease or condition.
 18. A composition comprising afirst nucleic acid sequence, wherein said first nucleic acid sequencecomprises an SBE nucleic acid sequence that binds a SEFIR domain of anACT1 protein, and wherein said SBE nucleic acid sequence comprises asequence shown in SEQ ID NO:1, 2, or 103, but which is not naturallyoccurring.
 19. The composition of claim 18, wherein said first nucleicacid sequence comprises modified bases to improve stability in vivo. 20.The composition of claim 18, wherein said first nucleic acid sequence ispresent in said composition at a level that is therapeutic whenadministered to a subject with an IL-17a related disease or condition.